Hydrogen peroxide and superoxide production by peripheral blood monocytes in leprosy.

Susceptibility to infection with Mycobacterium leprae, the causative organism of leprosy, is the result of a defect in cell-mediated immunity (CMI). The co-operation of macrophages and T lymphocytes is known to be essential for competent CMI response. In this study we have examined peripheral blood monocytes from a range of leprosy patients in an attempt to identify a possible defect in macrophage function. The ability of these cells to produce hydrogen peroxide and superoxide, two bactericidal metabolites of the monocyte/macrophage, has been measured. Monocytes from leprosy patients were found to be capable of producing normal amounts of hydrogen peroxide and superoxide, and no differences in production were found between tuberculoid, lepromatous and control monocytes. These results suggest that macrophages in leprosy are competent, and that probably a T lymphocyte defect contributes to susceptibility to this disease.     

Cellular effects of hematoporphyrin derivative photodynamic therapy on normal and neoplastic rat bladder cells.

HPD is known to localize in neoplastic cells and when exposed to the appropriate wavelength of light causes cytotoxicity. The authors have established a rat urothelial cell model for use in comparing and contrasting the effects of HPD photodynamic therapy (PDT) in normal (RBL-01) and transitional cell carcinoma (AY27) bladder cell lines. Uptake, toxicity, and morphologic damage following exposure to HPD PDT were evaluated. Trypan blue exclusion was used for determination of the toxicity of several HPD concentrations (1, 10, 25, and 50 micrograms/ml) with increasing duration of incubation with HPD (0, 1, 2, 4, 12, 24, and 48 hours). Both cell lines displayed increased toxicity with higher concentrations of HPD; however, the AY27 cells were more susceptible to the toxic effects of HPD PDT than the RBL-01 cells at the higher HPD doses studied (25 and 50 micrograms/ml). Viability decreased with increased duration of HPD incubation in RBL-01 cells up until 4 hours, after which it showed a steady increase. Viability decreased in the AY27 cells with increased duration of HPD incubation. An increase in serum concentration in the medium resulted in an increase in viability for both cell lines. Both cell lines demonstrated fast initial uptake of HPD followed by slower uptake over the time studied. By 24 and 48 hours the AY27 cells contained twice the amount of methanol-extractable porphyrins as the RBL-01 cells. The initial morphologic change following HPD PDT was damage to mitochondria. Mitochondrial damage occurred immediately after PDT in the AY27 cells and 30 minutes after PDT in the RBL-01 cells. Both cell lines exhibited a similar progression of cell injury; however, morphologic damage was observed earlier after PDT and appeared more extensive in the AY27 cells.     

Azurin of pathogenic Neisseria spp. is involved in defense against hydrogen peroxide and survival within cervical epithelial cells

Laz, a lipid-modified azurin of the human pathogens Neisseria gonorrhoeae and Neisseria meningitidis, is involved in defense against oxidative stress and copper toxicity; laz mutant strains are hypersensitive to hydrogen peroxide and copper. The N. gonorrhoeae laz mutant also has decreased survival in an ex vivo primary human ectocervical epithelial assay.     

Effect of mineral dust on generation of superoxide radicals and hydrogen peroxide by alveolar macrophages,granulocytes, and monocytes

Clinical Laboratory, Research Institute of Work Hygiene and Occupational Diseases, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. F. Izmerov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 110, No. 10, pp. 372–375, October, 1990.     

Hydrogen peroxide is produced by E. coli challenged haemocytes and regulates phagocytosis, in the medfly Ceratitis capitata. The active role of superoxide dismutase

Hydrogen peroxide (H₂O₂) participates as a second messenger in cell signaling. In this paper, the role of H₂O₂ was investigated, in Escherichia coli phagocytosis by the haemocytes of the medfly Ceratitis capitata. Block of H₂O₂ synthesis by specific enzymic inhibitors, namely N-ethylmaleimide (NEM) for NADPH oxidase and diethyldithiocarbamate (DDC) for SOD, resulted in the increase of E. coli phagocytosis. Immunoblot analysis, flow cytometry and confocal microscopy, revealed the constitutive expression of SOD, in the medfly haemocytes. Phagocytosis increased by small interfering RNA (siRNA) for SOD, revealing the active involvement of SOD and H₂O₂. Immunoblot analysis showed an increase of the ERK1/2 phosphorylation, in the presence of the above H₂O₂ synthesis enzymic inhibitors. In addition, confocal microscopy showed no co-localization of SOD with β integrin subunit. It appears that SOD participates in the regulation of bacterial phagocytosis, due to involvement of the produced H₂O₂ in the differential phosphorylation of MAP kinases.     

Growth hormone activation of human monocytes for superoxide production but not tumor necrosis factor production, cell adherence, or action against Mycobacterium tuberculosis.

We have previously demonstrated that growth hormone (GH) is a human macrophage-activating factor which primes monocytes for enhanced production of H2O2 in vitro. This report extends our observations to other monocyte functions relevant to infection. We find that GH also primes monocytes for O2- production, to a degree similar to the effect of gamma interferon. Neither macrophage-activating factor alone stimulates monocytes to release bioactive tumor necrosis factor. However, GH, unlike gamma interferon, does not synergize with endotoxin for enhanced tumor necrosis factor production. In further contrast, GH does not alter monocyte adherence or morphology, while phagocytosis and killing of Mycobacterium tuberculosis by GH-treated monocytes are also unaffected. Therefore, despite the multiplicity of the effects of GH on the immune system in vivo, its effects on human monocytes in vitro appear to be limited to priming for the release of reactive oxygen intermediates.     

Evidence for the role of superoxide radicals in neutrophil-mediated cytotoxicity.

Human peripheral neutrophils became cytotoxic to chicken red blood cells (CRBC) in the presence of lectins as assessed by release of 51chromium from labelled target cells. Phytohaemagglutinin (PHA) and concanavalin A (Con A), which caused time-dependent and dose-dependent cytotoxicity over a concentration range of 25--400 microgram/ml, also caused significant generation of superoxide radicals as measured by ferricytochrome C reduction. Pokeweed mitogen, which does not induce cytotoxicity over the same concentration range, was unable to promote superoxide generation by neutrophils. PHA-induced generation of superoxide paralleled and appeared to precede PHA-dependent cytotoxicity. Superoxide dismutase (SOD), which enzymatically destroys superoxide, caused moderate inhibition of PHA-dependent cytotoxicity over the concentration range of 100--500 microgram/ml whereas catalytically inactive enzyme had no effect. Incubation under oxygen-depleted conditions caused a marked decrease in both PHA-induced superoxide generation and cytotoxicity relative to that obtained with neutrophils incubated aerobically. These findings suggest a central role for superoxide radicals in causing target cell damage in this model of neutrophil-mediated cytotoxicity.     

Myeloperoxidase, hydrogen peroxide, chloride antimicrobial system: nitrogen-chlorine derivatives of bacterial components in bactericidal action against Escherichia coli.

In the presence of Escherichia coli, myeloperoxidase-catalyzed oxidation of chloride ion resulted in formation of long-lived chloramine and/or chloramide derivatives of bacterial components. The same amount of these nitrogen-chlorine (N-Cl) derivatives was obtained with either hypochlorous acid (HOCl) or the myeloperoxidase system, indicating that myeloperoxidase catalyzed the oxidation of chloride to HOCl. Identical killing was obtained with HOCl or the myeloperoxidase system. About 30 to 50% of the oxidizing equivalents of HOCl were detected as N-Cl derivatives of peptides or peptide fragments that were released from the bacteria. The apparent molecular weight distribution of the peptides decreased with increasing amounts of HOCl, suggesting that peptides were fragmented by oxidative cleavage of chloramide derivatives of peptide bonds. The remaining 50 to 70% of the oxidizing equivalents of HOCl were rapidly consumed in peptide bond cleavage or the oxidation of other bacterial components. There was a close correspondence between the oxidation of bacterial sulfhydryls and bactericidal action. The N-Cl derivatives were lost and the oxidation of bacterial sulfhydryls increased over a period of several h at 37 degrees C. These changes were accompanied by increased killing. The increase in sulfhydryl oxidation and killing could be prevented by washing the bacteria to remove the N-Cl derivatives. Therefore, the N-Cl derivatives could oxidize bacterial components long after the myeloperoxidase-catalyzed oxidation of chloride was complete.     

Transport of nitrullin,a new nitrosoalkylurea derivative,into tumor and normal cells

The interaction between the antitumor drug nitrullin and the system providing the transport ofl-lysine into P388 leukemic cells and murine enterocytes is studied. Two types of lysine carriers with low and high affinity for the substrate are identified. Nitrullin competitively inhibits the transport of³H-lysine and shows the same affinity for both carriers. It is similar to that of lysine for the low-affinity carrier and is 80-fold lower than lysine affinity for the high-affinity carrier. Kinetic characteristics of the low-affinity transport of³H-lysine and K_i of nitrullin are similar to those obtained at a reciprocal substrate-inhibitor ratio. Nitrullin does not inhibit active transport of³H-lysine into enterocytes against the background of considerable (70%) passive diffusion. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 12, pp. 648–650, December, 1996     

Hydrogen peroxide upregulates TNF-related apoptosis-inducing ligand (TRAIL) expression in human astroglial cells, and augments apoptosis of T cells

The brain is particularly vulnerable to oxygen free radicals, and these radicals have been implicated in the pathology of several neurological disorders. In this study, the modulation of TNF-related apoptosis-inducing ligand (TRAIL) expression by oxidative stress was shown in LN215 cells, an astroglioma cell line. Hydrogen peroxide (H2O2) treatment increased TRAIL expression in LN215 cells and H2O2-induced TRAIL augmented apoptosis in Peer cells, a cell line sensitive to TRAIL- mediated cell death. Our findings suggest that the upregulation of TRAIL in astroglial cells may abrogate immune cell effector functions.     

Interstitial cells of cajal are involved in neurotransmission in the gastrointestinal tract

Interstitial cells of Cajal (ICC) are important cells which coordinate gastrointestinal motility. ICC express Kit receptor tyrosine kinase, and Kit immunohistochemistry reveals ICC morphology and distribution in the gastrointestinal musculature. ICC show a highly branched morphology and form unique networks. Myenteric ICC (ICC-MY) are located at the layer of the myenteric plexus and serve as electrical pacemakers. Intramuscular ICC (ICC-IM) and ICC in the deep muscular plexus (ICC-DMP) are distributed within the muscular layers, and are densely innervated by excitatory and inhibitory enteric motor neurons and in close contact with nerve terminals. Recent studies combined with morphological and functional techniques directly revealed that ICC-IM and ICC-DMP are mediators of enteric motor neuro-transmission. These types of ICC express several receptors for neurotransmitters such as acetylcholine and substance P and show responses to excitatory nerve stimulations. ICC also express receptive mechanisms for nitric oxide, which is an inhibitory neurotransmitter in the gastrointestinal tract. They can respond to nitrergic nerve stimulation by cyclic GMP production. Kit mutant mice lack ICC-IM and show attenuated postsynaptic responses after intrinsic nerve stimulation. These findings indicate the importance for ICC in neurotransmission in the gastrointestinal tract.     

Mast cells and eosinophils are involved in activation of ulcerative colitis

PurposeThe intestinal mucosal immune cells such as the mast cells and eosinophils play an important role in the pathogenesis of ulcerative colitis (UC). The aim of present study was to compare the number of mast cells and eosinophils in patients with active and non-active ulcerative colitis. Another purpose was to found whether the number of eosinophils could correlate with number of mast cells in both tested groups.Material and MethodsThe twenty-five of formalin-fixed, paraffin-embedded tissue specimens of active ulcerative colitis, the twenty of non-active ulcerative colitis and the ten of controls were retrieved from archival material. Tryptase and chymase immunopositive cells were detected using immunohistochemical method. Additionally, the number of mast cells and eosinophils were detected using the most common histochemical methods.ResultsThe number of eosinophils and toluidine blue stained and tryptase immunopositive mast cells was significantly increased in active UC compared to non-active UC. In active stage of UC positive correlation between the number of mast cells stained with toluidine blue and the number of chymase and tryptase immunopositive mast cells were observed. Moreover, the number of eosinophils was significantly correlated with number of mast cells stained with toluidine blue and number of tryptase- and chymase immunopositive mast cells. In non-active stage of UC positive correlation was observed only between the number of mast cells stained with toluidine blue and chymase immunopositive cells and eosinophils.ConclusionsIn conclusion, our findings confirmed that mast cells and eosinophils are functionally involved in the course of UC.     

Trks are novel oncogenes involved in the induction of neovascularization, tumor progression, and nodal metastasis in oral squamous cell carcinoma

The function of tropomyosin receptor kinase (Trk) family including TrkA, TrkB, and TrkC in cancer remains unknown. The role of Trks in oral squamous cell carcinoma (OSCC) was examined. Knockdown of Trks provided inhibition of growth or invasion and decrease of apoptosis in OSCC cells, which expressed Trks at high levels. VEGF expression was associated with TrkA and TrkB expression; a decrease of VEGF-C and VEGF-D was observed in OSCC cells with TrkB knockdown. TrkC did not affect the expression of VEGF family. An immunohistochemical analysis of 102 OSCCs showed that TrkB expression was related to microvessel density (MVD), lymph vessel density (LVD), and poor prognosis. TrkC expression was correlated with clinical stage, lymph node metastasis, MVD, LVD, and poor prognosis. TrkA expression was associated with VEGF expression, whereas TrkB expression was associated with the expressions of VEGF, VEGF-C and VEGF-D. No significant association was found between the expression of TrkC and genes of the VEGF family. Expression of Trks was not associated with RUNX3 silencing by methylation in OSCC cells. Trks expression was inversely correlated with RUNX3 expression in the OSCC cases. These results suggested that Trks enhances progression of OSCC through angiogenesis and lymphangiogenesis.     

Guinea-pig alveolar macrophage killing of Mycobacterium tuberculosis, in vitro, does not require hydrogen peroxide or hydroxyl radical.

Alveolar macrophages from the lungs of guinea-pigs that had been vaccinated, boosted and then intravenously challenged with Mycobacterium bovis BCG, killed both a hydrogen-peroxide-resistant and a hydrogen-peroxide-sensitive strain of M. tuberculosis, in vitro. Pretreatment of the alveolar macrophages, in vitro, with catalase or mannitol, agents which remove hydrogen peroxide and the hydroxyl radical, respectively, did not decrease the extent of killing of either strain. In contrast, pretreatment of alveolar macrophages with catalase, reversed the inhibition of growth of Listeria monocytogenes.     

Cytoplasmic vacuoles of Rous virus transformed cells are organelles involved in cation uptake.

Cytoplasmic vacuoles induced during transformation of cells by Bryan strain Rous sarcoma virus (RSV-BH) have been studied using the cationic dye, neutral red(NR). Both the rate of uptake and the accumulation of NR are greater in RSV-BH transformed cells than non-transformed cells however, uptake was greater in vacuolated than in non-vacuolated cells, whether or not they were transformed. The NR was incorporated into pre-existing vacuoles in the absence of cytoplasmic staining, suggesting the existence of direct channels from the cell surface to the vacuoles. Other low mol. wt. cationic dyes could also be incorporated into vacuoles, although those with branched structures or cationic weights greater than 330 were excluded. No anionic dyes were incorporated. Infection of cells with a virus mutant, RSV-BH-Ta, induces temperature-dependent vacuolization. After a shift to the vacuole-permissive temperature, vacuoles developed at different rates and with morphological variations with different cations. Vacuoles which had formed in the presence of several cations, (K+, Rb+, tris+, choline+) failed to disappear when cells were incubated at a temperature sufficient to revert vacuoles formed in Na+-containing medium. No short-term effects of Cl-replacements (Br-, I-, or SO2-4) on vacuolization or reversal were observed. The results suggest that these vacuoles are organelles involved in cation uptake. A possible function for these organelles in RSV-BH induced malignancy is discussed.     

Antioxidants and ecto-5'-nucleotidase are not involved in the training-induced cardioprotection against ischaemia-reperfusion injury

Isolated Langendorff-perfused hearts from sedentary and prolonged (24 weeks) treadmill-trained rats were subjected to 30 min of normoxic perfusion either alone or followed by 20 min of global ischaemia, or by 20 min of global ischaemia and 15 min of normoxic reperfusion. Pre-ischaemic values of antioxidant enzyme activities and ecto-5'-nucleotidase activity were not different in sedentary and trained hearts but a 5-fold increase of 72-kDa heat shock protein (HSP72) levels was detected in trained myocardium. After ischaemia and reperfusion (I/R), metabolic recovery was better in trained than in sedentary hearts as indicated by higher ATP and creatine phosphate levels. However, antioxidant enzymatic activities, glutathione reductase, and total and mitochondrial superoxide dismutase decreased in trained rats after I/R, whereas they remained unchanged in the sedentary ones. Ecto-5'-nucleotidase activity was modified by I/R in sedentary as well as in trained hearts while HSP72 content did not change. Ecto-5'-nucleotidase activity and HSP72 content increased in parallel by the 30-min perfusion period. In conclusion, the cardioprotection induced by long-term training could be mediated by the exercise-induced increase in HSP72 levels and is not related to enhanced antioxidant systems or ecto-5'-NT activity.     

Induction of superficial bladder tumors in the female Fischer 344 rats with AY-27 tumor cells for the study of diffusion and localization of hemoglobin derived components (hematoporphyrin derivative) in view of photochemotherapy

Photochemotherapy (PCT) consists in administration of a photosensitizer and subsequent irradiation of the tumor with visible light. Routinely, the photosensitizer is given intravenously (i.v.), but the major drawback of this procedure is the resulting skin photosensitivity. The goal of our study is to examine whether intravesical (i.b.) instillation of the photosensitizer for PDT of bladder cancer might be feasible in order to target the tumors and to avoid the photosensitization phenomenon. After first studying the biodistribution of hematoporphyrin derivative (HpD) in vivo in the rat bladder, two and four hours after intravesical administration, by fluorescence microscopy, we compared two different methods for the induction of superficial bladder tumors in rats with AY-27 tumor cell line in order to perform the same study on bladder tumors. The best results for the penetration depth of HpD in the normal bladder wall were obtained two hours after the bladder instillation where the photosensitizer was detected only in the bladder surface (urothelium and small part of the chorion). That's why we must choose the most appropriate bladder tumor model in order to obtain superficial bladder tumors that mimic the clinical behavior of superficial bladder cancer in man. Both techniques used in this study gave a high tumor take rate in a short time (> 90%). But we really obtained superficial bladder tumors directly attached to the bladder surface with one of the two methods of tumor induction consisting in the abrasion of the bladder surface prior to the administration of the tumoral cells in the bladder cavity.