Reactive oxygen and nitrogen species in normal physiological processes

Reactive oxygen species (ROS) and reactive nitrogen species have generally been considered as being highly reactive and cytotoxic molecules. Besides their noxious effects, ROS participate in physiological processes in a carefully regulated manner. By way of example, microbicidal ROS are produced in professional phagocytes, ROS function as short-lived messengers having a role in signal transduction and, among other processes, participate in the synthesis of the iodothyronine hormones, reproduction, apoptosis and necrosis. Because of their ability to mediate a crosstalk between key molecules, their role might be dual (at least in some cases). The levels of ROS increase from a certain age, being associated with various diseases typical of senescence. The aim of this review is to summarize the recent findings on the physiological role of ROS. Other issues addressed are an increase in ROS levels during ageing, and the possibility of the physiological nature of this process.     

牛MBP诱导BALB/c和C57BL/6小鼠实验性变态性脑脊髓炎样反应的研究

为了进一步研究实验性变态反应性脑脊髓炎(EAE)动物模型,采用从新鲜牛脊髓中提取的MBP免疫诱导非敏感品系BALB/c小鼠和C57BL/6小鼠,通过对实验动物的症状、中枢神经系统的病理改变及细胞因子水平变化的检测,较成功地建立了C57BL/6、BALB/c小鼠的EAE模型,实验方法稳定可靠。     

Reactive oxygen and nitrogen species in sepsis-induced hepatic microvascular dysfunction

Objective and design

Hepatic microvascular dysfunction is a critical event in the development of liver failure during sepsis. Activated blood cells and reactive oxygen and nitrogen species (RONS) have been implicated in the pathogenesis of sepsis.

Methods

Intravital-videomicroscopy was used to determine whether RONS contribute to the recruitment of leukocytes/platelets in the hepatic microvasculature during sepsis. Six hours following cecal-ligation and puncture (CLP), disturbances of the hepatic microvasculature were assessed in WT-mice (C57Bl/6 J; n = 8), in mice lacking gp91^phox(n = 5), overexpressing superoxide-dismutase (SOD, n = 8), in WT-mice treated with a NOS-inhibitor (l-NAME, n = 5), lacking nNOS, eNOS or iNOS (n = 5 each), treated with the NO-donor DetaNO (n = 5), in WT-mice treated with gadolinium-chloride (GdCl₂, n = 5) and compared to a group of WT-mice following a sham operation (n = 8). Six hours post-CLP, the adhesion of leukocytes and platelets in terminal hepatic venules (THV) and sinusoids was quantified.

Results

In WT-mice, CLP elicited increases in the number of adherent leukocytes and platelets. Similar responses to CLP were noted in mice overexpressing SOD or lacking either eNOS or gp91^phox. The blood-cell recruitment was significantly blunted in septic iNOS-knockout mice and this response was reversed by pre-treatment with DetaNO.

Conclusion

These findings suggest that iNOS-derived NO is a determinant of the pro-inflammatory phenotype assumed by the hepatic microvasculature during sepsis.     

Airway inflammation and bronchial remodelling in toluene diisocyanate-exposed BALB/c mouse model

Toluene diisocyanate (TDI), a highly reactive industrial chemical, is one of the leading causes of occupation-related asthma in industrialized countries. The pathogenesis of TDI-induced asthma, however, remains not fully understood, in part due to lack of appropriate animal models. Twenty five female BALB/c mice (age: 8 weeks) were randomly divided into 5 groups: Ovabumin (OVA); OVA peptide amino acid residues No. 323-339 (Pep); TDI; alum and physiological saline. Mice were intraperitoneally injected with 25 microg OVA or pep absorbed on 300 microg alum, 300 microg alum or saline on days 0, 7 and 14. For the TDI group, mice were sensitized subcutaneously with 20 microl neat TDI on day 0; 20 microl of TDI in olive oil (1:10) on days 7 and 14; on days 21-23. Then each group was challenged intranasally with 20 microl of 1% OVA, 1% Pep, 1% TDI, 10% alum and saline respectively. On day 28, mice were killed under pentothal anesthesia. The results demonstrated that neutrophil-dominant inflammation with a few eosinophil infiltration occurred in the peri-bronchial and peri-vascular regions of the lungs. This was accompanied by hyperplasia/hypertrophy of cells lining the airways and mucus production as shown by HE staining. Positive immunohistochemical MBP staining in parenchyma was also shown. Th2 cytokine IL-4 and IgE production were significant increased 5 days after last challenge while IFN-gamma level was below the detection limit. Conclusion: the clear elevation of IL-4 and IgE could allow to conclude a possible Th2-like dominated allergic response in TDI-exposed BALB/c mouse model.     

Efficacy of a Proteus mirabilis outer membrane protein vaccine in preventing experimental Proteus pyelonephritis in a BALB/c mouse model.

A BALB/c mouse model of nonobstructive, ascending Proteus mirabilis pyelonephritis was characterized bacteriologically, histologically, and serologically from 3 to 28 days. Intravesicular administration of 2 X 10(8) P. mirabilis K7 resulted in the septic death of 9 (16%) of 57 mice by day 15. Among the survivors, K7 colonized the kidneys in great numbers until day 21. Histological examination of the kidneys revealed acute inflammation which was characterized by neutrophil infiltration by day 3, renal necrosis by day 7, and fibroblastic infiltration by day 14 which persisted at least until day 28. The immunoglobulin G response to the outer membrane proteins (OMP) was assessed by enzyme-linked immunosorbent assay and Western blotting (immunoblotting). Anti-OMP immunoglobulin G antibodies were detected as early as day 7, and the reciprocals of their titers rose progressively up to day 28 (i.e., greater than or equal to 500). This model was also used to assess the efficacy of OMP and lipopolysaccharide (LPS) immunization in preventing renal infection. K7 OMP or LPS (100 micrograms) preparations were administered intramuscularly in Freund's complete adjuvant. After 2 weeks, mice were intravesicularly challenged with 2 X 10(8) bacteria of the homologous K7 strain or one of four heterologous strains. Compared with the saline-immunized control group and K7 LPS-immunized mice, K7 OMP recipients were protected from death when challenged by homologous or heterologous strains. In addition, K7 OMP recipients were protected (P less than 0.003) from subsequent renal infection when challenged by the K7 strain and had more rapid bacterial renal clearance when challenged by three of four heterologous strains. OMP recipients produced antibodies which bound major OMP moieties (viz., 36- to 39-kDa cell wall constituents) as assessed by Western blotting. These results support the concept that immunization with selected bacterial protein surface coat constituents can prevent uromucosal infection by interfering with colonization or renal injury.     

A novel and effective approach of developing aggressive experimental autoimmune gastritis in neonatal thymectomized BALB/c mouse by polyinosinic:polycytidylic acid

Neonatal thymectomy induces autoimmune gastritis (AIG) in 40-70% of BALB/c mice. We presumed that induction of autoimmunity by polyinosinic:polycytidylic acid (poly I:C) might allow development of a more aggressive model of AIG. A group of BALB/c mice were thymectomized on day 3 after birth. Neonatal thymectomized mice were either injected with poly I:C or phosphate-buffered saline (PBS). Non thymectomized neonatal BALB/c mice were injected with only poly I:C. All neonatal thymectomized mice injected with poly I:C developed 3 cardinal features of AIG: (1) moderate to severe degree gastritis (2) presence of autoantibody to H(+)/K(+) ATPase and (3) loss of parietal cells. However, only 70% of the PBS-treated neonatal thymectomized BALB/c mice developed some, but not, all features of AIG. A mild degree of AIG was seen in 12 of 31 nonthymectomized BALB/c mice administered with only poly I:C. Administration of poly I:C in neonatal thymectomized BALB/c mice in the first and second week appeared to be the most effective for induction of aggressive AIG. The levels of interleukin (IL)-6, IL-12p70, interferon-gamma and tumour necrosis factor-alpha were significantly higher in poly I:C-injected thymectomized mice compared to PBS-injected neonatal thymectomized mice (P < 0.05). The frequencies of CD4(+)CD25(+) regulatory T cells in the spleen were significantly decreased in neonatal thymectomized mice administered with poly I:C compared to PBS-treated neonatal thymectomized mice (P < 0.01). Taken together, these results suggest that induction of inflammatory cytokines and reduction of regulatory T cells by poly I:C might contribute to the development of an aggressive model of AIG in neonatal thymectomized BALB/c mice.     

Reactive oxygen and nitrogen species: implications for cardiovascular device engineering

The development of medical devices for cardiovascular applications has suffered due to lack of understanding of why vascular wall cells act nonphysiologically when exposed to biomaterials. One possible reason might be the chemical environment associated with cardiovascular disease. An improved understanding of cellular and subcellular mechanisms may assist in future device design to account for the disease environment. Reactive oxygen and nitrogen species (ROS/RNS) are produced through normal cellular metabolism and are rendered harmless by enzymatic systems. However, during a disease process, these systems may act aberrantly, and either fail to convert ROS and RNS to harmless substances or by producing more oxidants. There is indirect evidence that the implantation of biomedical materials may also be responsible for the triggering of these aberrant pathways that may lead to the eventual failure of the device. The understanding of how the vascular environment may be changed at the subcellular level by the presence of a biomaterial is critical. In the following pages, we hope to review the current thinking within vascular biology regarding ROS and RNS, how they are measured, and how they may impact vascular cells.     

A new model of busulphan-induced chronic bone marrow aplasia in the female BALB/c mouse

Aplastic anaemia (AA) is characterized by hypocellular marrow, pancytopenia, and risk of severe anaemia, haemorrhage and infection. AA is often idiopathic, but frequently occurs after exposure to drugs/chemicals. However, the pathogenesis of AA is not clearly understood, and there are no convenient animal models of drug-induced AA. We have evaluated regimens of busulphan (BU) administration in the mouse to produce a model of chronic bone marrow aplasia showing features of human AA. Mice were given 8 doses of BU at 0, 5.25 and 10.50 mg/kg over 23 days; marrow and blood samples were examined at 1, 19, 49, 91 and 112 days after dosing. At day 1 post dosing, in mice treated at 10.50 mg/kg, nucleated marrow cells, CFU-GM and Erythroid-CFU were reduced. Similarly, peripheral blood erythrocytes, leucocytes, platelets and reticulocytes were reduced. At day 19 and 49 post dosing, there was a trend for parameters to return towards normal. However, at day 91 and 112 post dosing, values remained significantly depressed, with a stabilized chronic bone marrow aplasia. At day 91 and 112 post dosing, marrow cell counts, CFU-GM and Erythroid-CFU were decreased; marrow nucleated cell apoptosis and c-kit+ cell apoptosis were increased; peripheral blood erythrocyte, leucocyte, and platelet counts were reduced. We conclude that this is a model of chronic bone marrow aplasia which has many interesting features of AA. The model is convenient to use and has potential in several areas, particularly for investigations on mechanisms of AA pathogenesis in man.     

Pathogenesis of defined invasion mutants of Yersinia enterocolitica in a BALB/c mouse model of infection.

It has been hypothesized for many years that the ability of Yersinia spp. to invade tissue culture cells is reflective of their ability to penetrate the intestinal epithelium and that this capacity is an important aspect of the disease process. Three different genes from Yersinia spp. that are involved in the tissue culture invasion phenotype have been identified: inv, ail, and yadA. It was previously shown that inv is necessary for efficient penetration of the intestinal epithelium by Yersinia enterocolitica. The present study was initiated to determine whether other known Yersinia invasion factors could promote uptake of the bacteria by mice in the absence of invasion. In addition, the roles of these three invasion factors in the survival of the bacteria, lethality for mice, and development of pathology were compared. We found that YadA is necessary for persistence of Y. enterocolitica in Peyer's patches, and consistent with this observation, the yadA mutant was avirulent for mice infected either orally or intraperitoneally. In addition, the inv yadA double mutant was avirulent. Histological and immunohistological examination of the Peyer's patches of infected mice indicated that despite the presence of large numbers of CFU at 24 h the yadA and ail yadA mutants cause only minimal pathology and recruitment of macrophages. At 42 h postinfection, Peyer's patches from mice infected with the inv mutant showed no pathology, despite the prediction that some of the mice by this time would be colonized. However, at 72 h, inflammation and necrosis were evident in some Peyer's patches. Together, these observations suggest that for visible pathology to develop, a threshold number of bacteria (> 10(5)) is needed and the bacteria need to persist for more than 24 h. Lastly, YadA but not Ail may play a role in the less efficient, delayed invasion of the intestinal epithelium observed for the inv mutant.     

Pathogenesis of experimental Ebola Zaire virus infection in BALB/c mice.

Guinea-pigs and non-human primates have traditionally been used as animal models for studying Ebola Zaire virus (EBO-Z) infections. The virus was also recently adapted to the stage of lethal virulence in BALB/c mice. This murine model is now in use for testing antiviral medications and vaccines. However, the pathological features of EBO-Z infection in mice have not yet been fully described. To identify sites of viral replication and characterize sequential morphological changes in BALB/c mice, adult female mice were infected with mouse-adapted EBO-Z and killed in groups each day for 5 days post-infection. Tissues were examined by light microscopy, immunohistochemistry, electron microscopy and in-situ hybridization. As in guinea-pigs and non-human primates, cells of the mononuclear phagocytic system were the earliest targets of infection. Viral replication was observed by day 2 in macrophages in lymph nodes and spleen. By the time of onset of illness and weight loss (day 3), the infection had spread to hepatocytes and adrenal cortical cells, and to macrophages and fibroblast-like cells in many organs. Severe lymphocytolysis was observed in the spleen, lymph nodes and thymus. There was minimal infection of endothelial cells. All of these changes resembled those observed in EBO-Z-infected guinea-pigs and non-human primates. In contrast to the other animal models, however, there was little fibrin deposition in the late stage of disease. The availability of immunodeficient, "gene-knockout" and transgenic mice will make the mouse model particularly useful for studying the early steps of Ebola pathogenesis.     

Protection against infertility in a BALB/c mouse salpingitis model by intranasal immunization with the mouse pneumonitis biovar of Chlamydia trachomatis.

Female BALB/c mice were immunized intranasally with the mouse pneumonitis biovar of Chlamydia trachomatis and subsequently challenged in the ovarian bursa (C. trachomatis immunized, C. trachomatis challenged). Two groups of mice served as controls. One group was sham immunized intranasally with mock-infected HeLa 229 cell extracts and was challenged in the ovarian bursa with C. trachomatis MoPn (sham immunized, C. trachomatis challenged). The second control group was sham immunized and not challenged (sham immunized, nonchallenged). Before challenge, the C. trachomatis-immunized, C. trachomatis-challenged animals mounted a significant humoral response as shown by high immunoglobulin G (IgG), IgM, and IgA levels and high levels of neutralizing antibodies in serum and moderate IgG and IgA titers in vaginal secretions. Reactivity by Western blot (immunoblot) to the lipopolysaccharide, 30-, 40- (major outer membrane protein), and 60-kDa cysteine-rich proteins and 75- and 100-kDa chlamydial components could be demonstrated. However, reactivity to the 60-kDa heat shock protein was only observed 22 days after challenge. In addition, this group of animals mounted a significant immune response to chlamydial antigens, as shown by a lymphocyte proliferation assay, compared with the sham-immunized nonchallenged mice. After intrabursal challenge, there was no C. trachomatis shedding from the vagina in the C. trachomatis-immunized, C. trachomatis-challenged animals, while 63% of the sham-immunized, C. trachomatis-challenged mice had a positive C. trachomatis culture. In addition, histological sections from the genital tract showed, at 2 weeks postchallenge, a marked acute inflammatory reaction in the sham-immunized, C. trachomatis-challenged animals while in the C. trachomatis-immunized, C. trachomatis-challenged mice there was minimal inflammatory reaction. When the animals were mated, only 12% of the mice from the sham-immunized, C. trachomatis-challenged mice were fertile. In contrast, 94 and 80% of the sham-immunized, nonchallenged and C. trachomatis-immunized, C. trachomatis-challenged mice, respectively, were fertile.     

A novel model of drug hapten-induced hepatitis with increased mast cells in the BALB/c mouse

Clinical evidence suggests that idiosyncratic hepatitis following administration of halogenated volatile anesthetics is mediated by autoimmune responses. No murine model to study mechanisms of anesthetic-induced or any other form of drug-induced idiosyncratic hepatitis exists. Anesthetics are believed to trigger hepatitis by covalently linking a trifluoroacetyl (TFA) chloride hapten to hepatic proteins, forming haptenated self-proteins. To test this hypothesis, we developed a hapten-induced model of hepatitis by immunization with syngeneic S100 liver proteins covalently coupled to TFA (TFA-S100). We found that TFA-S100 induced hepatitis was more severe than disease induced by S100 plus adjuvants or by the adjuvant alone and was characterized by neutrophil, mast cell, and eosinophil infiltration. TFA-specific IgG1 antibodies directly correlated with hepatitis, whereas S100 autoantibodies did not. TNF-alpha, IL-1beta, and IL-6 released from splenocytes collected 2 weeks after TFA-S100 inoculation were increased resembling the elevated serum cytokines reported in patients with autoimmune hepatitis (AIH). Three weeks after inoculation, the peak of hepatitis, we noted decreased numbers of Kupffer cells and lower levels of IL-6 and IL-10 in the liver, cytokines produced by Kupffer cells. This is the first report, to our knowledge, of a hapten-induced model of hepatitis with immune and autoimmune features.     

Reactive oxygen and nitrogen species and cellular and organismal decline: amelioration with melatonin

Cellular and organismal decline is, in part, believed to be a consequence of oxygen and nitrogen-based reactants which persistently damage macromolecules throughout a lifetime. The resulting accumulation of damaged molecules eventually seriously compromises essential functions of cells leading to their death. Excessive cellular loss causes deterioration of organ function and inevitably to the demise of the organism. The sequence of events, known as the free radical theory of aging, is widely espoused by biological gerontologists. Antioxidants are commonly employed to combat molecular damage mediated by oxygen and nitrogen-based reactants. One of these protective agents is melatonin. Melatonin has several distinct advantages as a preserver of organelle structure and function. It is widely distributed in organisms and within cells. It works via a number of mechanisms to reduce oxidative damage. Thus, melatonin scavenges a number of reactants including the hydroxyl radical (*OH), hydrogen peroxide (H(2)O(2)), nitric acid (NO*), peroxynitrite (ONOO(-)) and peroxynitrous acid (ONOOH). One of the products of melatonin's interaction with H(2)O(2), i.e., N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK), is also a highly efficient radical scavenger. The cascade of reactions where the secondary metabolites are also effective scavenges is believed to contribute to melatonin's high efficacy in reducing oxidative damage. Besides its direct scavenging actions, melatonin stimulates several antioxidative enzymes including superoxide dismutase, glutathione peroxidase and glutathione reductase in addition to inhibiting a proxidative enzyme, nitric oxide synthase. This combination of actions assists melatonin in protecting cells from the degenerative changes normally associated with aging and age-related diseases.     

Immune response to Nocardia brasiliensis antigens in an experimental model of actinomycetoma in BALB/c mice

Nine- to twelve-week-old BALB/c mice were injected in footpads with 10(7) CFU of a Nocardia brasiliensis cell suspension. Typical actinomycetoma lesions, characterized by severe local inflammation with abscess and fistula formation, were fully established by day 28 after infection. These changes presented for 90 days, and then tissue repair with scar formation slowly appeared, with complete healing after 150 days of infection. Some animals developed bone destruction in the affected area. Histopathology showed an intense inflammatory response, with polymorphonuclear cells and hyaloid material around the colonies of the bacteria, some of which were discharged from draining abscesses. Sera from experimental animals were analyzed by Western blotting, and immunodominant antigens P61 and P24 were found as major targets for antibody response. Anti-P24 immunoglobulin M (IgM) isotype antibodies were present as early as 7 days, IgG peaking 45 days after infection. Lymphocyte proliferation with spleen and popliteal lymph node cells demonstrated thymidine incorporation at 7 days after infection, the stimulation index decreasing by day 60. Levels of interleukin-1 (IL-1), IL-2, IL-4, IL-6, tumor necrosis factor alpha, and gamma interferon (IFN-gamma) were determined by enzyme-linked immunosorbent assay in the sera of infected animals. The circulating levels of IFN-gamma increased more than 10 times the basal levels; levels of IL-4, IL-6 and IL-10 also increased during the first 4 days of infection.     

苯甲酸雌二醇对BALB/c小鼠系统性红斑狼疮模型诱导的影响

目的：探讨不同剂量的苯甲酸雌二醇（Estradiol benzoate）对BALB/c小鼠系统性红斑狼疮（Systemic lupusery-thematosus，SLE）模型诱导的影响。方法：BALB/c小鼠卵巢摘除后用同系小鼠ConA活化的脾细胞（5×107）免疫3次，同时肌注不同剂量苯甲酸雌二醇，6周后以ELISA法检测血清抗核抗体（ANA）、抗组蛋白抗体（AHA）及血清和肾组织匀浆雌二醇（Estradiol，E2）水平，H.E、PAS染色观察肾脏病理改变，透射电镜检查肾脏超微结构的改变，直接荧光法检查IgG类免疫复合物的沉积。结果：经同系小鼠ConA活化的脾细胞免疫的BALB/c小鼠均发生红斑狼疮样改变，但不同剂量苯甲酸雌二醇组小鼠的自身抗体的产生及肾脏病理损害差异显著（P〈0.05）。结论：机体不同的雌二醇水平与SLE的病变程度相关。     

Modulation of bleomycin-induced pulmonary fibrosis in the BALB/c mouse by cyclophosphamide-sensitive T cells.

Endotracheal bleomycin treatment is an effective inducer of pneumonitis and pulmonary fibrosis. Certain strains of mice, however, develop only minimal or no pulmonary fibrosis after treatment with bleomycin. The mechanism of unresponsiveness or low responsiveness in the BALB/c strain of mice is examined in this article. Pretreatment with cyclophosphamide (100 mg/kg) 2 days prior to bleomycin instillation significantly augmented the fibrotic response in these mice. Treatment by cyclophosphamide alone at the same dosage caused no significant pulmonary disease or fibrosis. Furthermore, suppression of fibrosis in the cyclophosphamide-pretreated animals could be reconstituted with spleen cells from normal untreated donor mice. If the donor spleen cells were depleted of T cells by cytotoxic anti-Thy-1.2 antisera prior to infusion into recipient animals, no such reconstitution was observed, suggesting that a population of splenic T cells was responsible for the effect. Since this dose of cyclophosphamide is known to be cytotoxic for suppressor T cells, these data would signify that the intensity of the lung fibrogenic response to bleomycin in BALB/c mice can be modulated by a population of this T cell subset. Furthermore, the cell reconstitution data would exclude the possibility that cyclophosphamide augments the bleomycin response by non-T-cell-mediated synergistic effects on lung injury. Nevertheless, the results conclusively demonstrate the ability of T cells to modulate pulmonary fibrosis in vivo, at least in the BALB/c mouse.