Planting a chemical flag on antigens.

Next-generation live vaccines are created by autonomous production of nitrated antigens.

Effects of phosphodiesterase 5 inhibitor sildenafil on the respiratory parameters, inflammation and apoptosis in a saline lavage-induced model of acute lung injury.

Acute lung injury (ALI) is associated with deterioration of alveolar-capillary lining and transmigration and activation of inflammatory cells. Sildenafil, phosphodiesterase 5 (PDE5) inhibitor, inhibits degradation of cyclic guanosine monophosphate (cGMP) by competing with cGMP for binding site of PDE5. Positive effects of sildenafil treatment result from influencing proliferation of regulatory T cells and production of proinflammatory cytokines and autoantibodies as well as from modulation of platelet activation, angiogenesis, and pulmonary vasoreactivity. This study evaluated if intravenous sildenafil can influence inflammation, edema formation, apoptosis, and respiratory parameters in rabbits with a model of ALI induced by repetitive lung lavage by saline (30 ml/kg). animals were divided into 3 groups: ALI without therapy (ALI), ALI treated with sildenafil intravenously (1 mg/kg; ALI + Sil), and healthy ventilated controls (Control) which were oxygen-ventilated for 4 hours following treatment administration. during this period, respiratory parameters (ventilator pressures, lung compliance, blood gases, oxygenation indexes etc.) were regularly measured. at the end of experiment, animals were overdosed by anesthetics. The left lung was saline-lavaged and total and differential cell counts and protein content in the bronchoalveolar lavage fluid (BAL) were estimated. The right lung was used for determination of lung edema formation expressed as wet/dry lung weight ratio, for detection of inflammation and oxidative stress markers by ELISA methods, and for detection of lung epithelial cells apoptosis by TUNEL methods and level of caspase-3. Sildenafil treatment reduced leak of cells (P < 0.05), particularly of neutrophils (P < 0.001) into the lung, release of pro-inflammatory mediators (TNF-α, P < 0.001; IL-8 and IL-6, P < 0.01), level of nitrite/nitrate (P < 0.001), markers of oxidative damage (3-nitrotyrosine and malondialdehyde, both P < 0.01), lung edema formation (P < 0.01), protein content in BAL (P < 0.001), and apoptosis of epithelial cells (P < 0.01), and improved respiratory parameters. Concluding, the results indicate a future potential of PDE5 inhibitors also for the therapy of ALI.

Nitration of MOG diminishes its encephalitogenicity depending on MHC haplotype.

Post-translational modifications of autoantigens are hypothesized to affect their immunogenicity. We here report that nitration of tyrosine 40 in Myelin Oligodendrocyte Glycoprotein (MOG) abrogates its encephalitogenicity both at protein and peptide levels in the experimental autoimmune encephalomyelitis (EAE) model in H2b C57BL/6 mice. Furthermore, nitrated MOG displays inferior antigen-specific proliferation of 2D2 splenocytes in vitro. Conversely, H2q DBA1 mice remain fully susceptible to EAE induction using nitrated MOG as the dominant epitope of H2q mice is unaltered. Molecular modeling analysis of the MOG35-55/H2-IAb complex and bioinformatics peptide binding predictions indicate that the lack of T cell reactivity towards nitrated MOG can be attributed to the inability of murine H2-IAb to efficiently present the altered peptide ligand of MOG35-55 because the nitrated tyrosine 40 cannot be accommodated in the p1 anchor pocket. In conclusion we demonstrate nitration as a relevant determinant affecting T cell recognition of carrier antigen depending on MHC haplotype. Our data have implications for understanding the role of post-translationally modified antigen in autoimmunity.

The equine alveolar macrophage: functional and phenotypic comparisons with peritoneal macrophages.

Alveolar macrophages (AMs) constitute the first line of defence in the lung of all species, playing a crucial role in the regulation of immune responses to inhaled pathogens. A detailed understanding of the function and phenotype of AMs is a necessary pre-requisite to both elucidating their role in preventing opportunistic bacterial colonisation of the lower respiratory tract and developing appropriate preventative strategies. The purpose of the study was to characterise this important innate immune cell at the tissue level by making functional and phenotypic comparisons with peritoneal macrophages (PMs). We hypothesised that the tissue of origin determines a unique phenotype of AMs, which may constitute an appropriate therapeutic target for certain equine respiratory diseases. Macrophages isolated from the lung and the peritoneal cavity of 9 horses were stimulated with various toll like receptor (TLR) ligands and the production of nitrite, tumour necrosis factor alpha (TNFα), interleukin (IL) 10 and indoleamine 2,3-dioxygenase (IDO) were measured by the Griess reaction and enzyme linked immunosorbent assay (ELISA) and/or quantitative polymerase chain reaction, respectively. Cells were also compared on the basis of phagocytic-capacity and the expression of several cell surface markers. AMs, but not PMs, demonstrated increased TNFα release following stimulation with LPS, polyinosinic polycytidylic acid (Poly IC) and heat-killed Salmonella typhinurium and increased TNFα and IDO mRNA expression when stimulated with LPS. AMs showed high expression of the specific macrophage markers cluster of differentiation (CD) 14, CD163 and TLR4, whereas PMs showed high expression of TLR4 only. AMs, but not PMs, demonstrated efficient phagocytic activity. Our results demonstrate that AMs are more active than PMs when stimulated with various pro-inflammatory ligands, thus supporting the importance of the local microenvironment in the activation status of the macrophage. This information provides a valuable knowledge base on which to improve our understanding of the role of macrophages and their microenvironment in equine innate immunity.

Distinct pattern of immunophenotypic features of innate and adaptive immunity as a putative signature of clinical and laboratorial status of patients with localized cutaneous leishmaniasis.

In this study, we have analysed the phenotypic features of innate/adaptive immunity of patients with localized cutaneous leishmaniasis (LCL), categorized according to their clinical/laboratorial status, including number of lesion (L1; L2­4), days of illness duration (≤60;>60) and positivity in the Montenegro skin test (MT−;MT+). Our findings highlighted a range of phenotypic features observed in patients with LCL (↑%HLA-DR+ neutrophils; ↑CD8+ HLA-DR+/CD4+ HLA-DR+ T cell ratio; ↑HLA-DR in B lymphocytes, ↑%CD23+ neutrophils, monocytes and B cells; ↑α-Leishmania IgG and ↑serum NO2⁻ + NO3⁻). Selective changes were observed in L1 (↑%HLA-DR+ neutrophils, ↑CD8+ HLA-DR+/CD4+ HLA-DR+ T cell ratio and ↑serum NO2⁻ + NO3⁻) as compared to L2­4 (↑%CD5− B cells; ↑CD23+ B cells and ↑α-Leishmania IgG). Whilst ≤60 presented a mixed profile of innate/adaptive immunity (↓%CD28+ neutrophils and ↑%CD4+ T cells), >60 showed a well-known leishmanicidal events (↑CD8+ T cells; ↑serum NO2⁻ + NO3⁻ and ↑α-Leishmania IgG). MT+ patients showed increased putative leishmanicidal capacity (↑%HLA-DR+ neutrophils; ↑%CD23+ monocytes; ↑CD8+ HLA-DR+/CD4+ HLA-DR+ T cell ratio and ↑ serum NO2⁻ + NO3⁻). Overall, a range of immunological biomarkers illustrates the complex immunological network associated with distinct clinical/laboratorial features of LCL with applicability in clinical studies.

Postvaccinal changes in the nitric oxide system after immunization with live dry tularemia vaccine.

Immunization of outbred male albino mice with live dry tularemia vaccine in a dose of 50 CFU/mouse was associated with stimulation of the NO system and accumulation of NO metabolites (nitrites and nitrates) in splenic and hepatic tissues. High levels of these metabolites persisted by day 14 after the initial and repeated immunization. These results suggest that the immunotropic effect of live dry tularemia vaccine manifested by not only modulation of the functions of immunocompetent T and B cells, NK and K cells, micro- and macrophages, but also by stimulation of intracellular anti-infection defense at the tissue level via intensification of NO synthesis.

Autocatalytic tyrosine nitration of prostaglandin endoperoxide synthase-2 in LPS-stimulated RAW 264.7 macrophages.

In the literature, biological tyrosine nitrations have been reported to depend not only on peroxynitrite but also on nitrite/hydrogen peroxide linked to catalysis by myeloperoxidase. In endotoxin-stimulated RAW 264.7 macrophages, we have detected a major nitrotyrosine positive protein band around 72 kDa and identified it as prostaglandin endoperoxide synthase-2 (PGHS-2). Isolated PGHS-2 in absence of its substrate arachidonate was not only tyrosine-nitrated with peroxynitrite, but also with nitrite/hydrogen peroxide in complete absence of myeloperoxidase. Our data favor an autocatalytic activation of nitrite by PGHS-2 with a subsequent nitration of the essential tyrosine residue in the cyclooxygenase domain. Under inflammatory conditions, nitrite formed via NO-synthase-2 may therefore act as an endogenous regulator for PGHS-2 in stimulated macrophages. Nitration of PGHS-2 by the autocatalytic activation of nitrite further depends on the intracellular concentration of arachidonate since arachidonate reacted competitively with nitrite and could prevent PGHS-2 from nitration when excessively present.

Nitric oxide metabolite determinations reveal continuous inflammation in multiple sclerosis.

Nitric oxide (NO) is formed as a consequence of induction of the iNOS enzyme during inflammatory disorders. To investigate NO production in multiple sclerosis (MS), we determined the concentrations of its oxidation products (NOx) in the cerebrospinal fluid (CSF) and plasma of 61 MS patients. The patients were divided into three groups on the basis of their clinical disease activity. The total levels of NOx in CSF were significantly increased in all MS groups as compared to healthy controls and tension headache patients. CSF nitrite correlated with clinical disease activity. At exacerbation, the CSF nitrite levels exceed the plasma level. This suggests that clinical disease activity is due to a CNS inflammatory response, which is more intense and qualitatively different from that during clinical stable phases. This study supports NO involvement in the pathogenesis of MS and determination of nitrite levels may be useful a surrogate marker for disease activity.

Antibody-dependent cell-mediated cytotoxicity to newly excysted juvenile Fasciola hepatica in vitro is mediated by reactive nitrogen intermediates.

Passive intraperitoneal transfer of sera from Fasciola hepatica-infected sheep, cattle or rats can protect naive rats from F. hepatica infection, suggesting a parasite killing mechanism within the peritoneal cavity that is dependent on the presence of parasite-specific antibody. We investigated antibody-dependent cell-mediated cytotoxicity by resident peritoneal lavage cell populations, containing large numbers of monocytes/macrophages, as a potential host resistance mechanism by which juvenile flukes could be killed within the peritoneal cavity of naive rats. Comparative studies were conducted using cell populations containing large numbers of monocytes/macrophages from sheep. The results demonstrate that monocyte/macrophage-rich lavage cell populations from rat and sheep differ substantially in their ability to generate nitric oxide. Only resident rat peritoneal lavage cells were able to mediate antibody-dependent cell-mediated cytotoxicity against newly excysted juvenile liver fluke. The mechanism of cytotoxicity was dependent on, and directly proportional to, the production of nitric oxide and required attachment of effector cells to the newly excysted juvenile liver fluke tegument, which occurred following the addition of sera from F. hepatica-infected animals. This is the first report demonstrating a mechanism of cell-mediated cytotoxicity to newly excysted juvenile liver fluke.

Western blot analysis for nitrotyrosine protein adducts in livers of saline-treated and acetaminophen-treated mice.

The hepatic centrilobular necrosis produced by the analgesic/antipyretic acetaminophen correlates with metabolic activation of the drug leading to its covalent binding to protein. However, the molecular mechanism of the toxicity is not known. Recent immunohistochemical analyses using an antinitrotyrosine antiserum indicated that nitrotyrosine protein adducts co-localized with the acetaminophen-protein adducts in the centrilobular cells of the liver. Nitration of proteins is believed to occur by peroxynitrite, a substance formed by the rapid reaction of superoxide with nitric oxide. Nitric oxide and superoxide may be formed by activated Kupffer cells or by other cells. Because we were unable to successfully utilize the commercial antiserum in Western blot analyses of liver fractions, we developed a new antiserum. With our antiserum, liver fractions from saline-treated control and acetaminophen-treated mice were successfully analyzed for nitrated proteins. The immunogen for this new antiserum was synthesized by coupling 3-nitro-4-hydroxybenzoic acid to keyhole limpet hemocyanin. A rabbit immunized with this adduct yielded a high titer of an antiserum that recognized BSA nitrated with peroxynitrite. Immunoblot analysis of nitrated BSA indicated that nitrotyrosine present in a protein sample could be easily detected at levels of 20 pmoles. Immunohistochemical analyses indicated that nitrotyrosine protein adducts were detectable in the centrilobular areas of the liver. Immunoblot analysis of liver homogenates from both saline-treated and acetaminophen-treated mice (300 mg/kg) indicate that the major nitrotyrosine protein adducts produced have molecular weights of 36 kDa, 44 kDa, and 85 kDa. The 85-kDa protein stained with the most intensity. The hepatic homogenates of the acetaminophen- treated mice showed significantly increased levels of all protein adducts.

Protective effect of poly(ADP-ribose) synthetase inhibition on multiple organ failure after zymosan-induced peritonitis in the rat.

BACKGROUND AND METHODS: In the present study, we tested the hypothesis that peroxynitrite and subsequent activation of the nuclear enzyme poly(ADP-ribose) synthetase (PARS) play a role in the pathogenesis of multiple organ failure induced by peritoneal injection of zymosan in the rat. Animals were randomly divided into six groups (ten rats for each group). The first group was treated with ip administration of saline solution (0.9% NaCl) and served as the sham group. The second group was treated with ip administration of zymosan (500 mg/kg suspended in saline solution). In the third and fourth groups, rats received ip administration of 3-aminobenzamide (10 mg/kg) 1 and 6 hrs after zymosan or saline administration, respectively. In the fifth and sixth groups, rats received ip administration of nicotinamide (50 mg/kg) 1 and 6 hrs after zymosan or saline administration, respectively. After zymosan or saline injection, animals were monitored for 72 hrs to evaluate systemic toxicity (conjunctivitis, ruffled fur, diarrhea, and lethargy), loss of body weight, and mortality. RESULTS: A severe inflammatory response, characterized by peritoneal exudation, high plasma and peritoneal levels of nitrate/nitrite (the breakdown products of nitric oxide), and leukocyte infiltration into peritoneal exudate, was induced by zymosan administration. This inflammatory process coincided with the damage of lung, small intestine, and liver as assessed by histologic examination and by an increase of myeloperoxidase activity, which is indicative of neutrophil infiltration. Zymosan-treated rats showed signs of systemic illness, significant loss of body weight, and high mortality rates. Peritoneal administration of zymosan in the rat also induced a significant increase in the plasma levels of peroxynitrite as measured by the oxidation of the fluorescent dihydrorhodamine 123. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine, a specific "footprint" of peroxynitrite, in the lung of zymosan-shocked rats. In vivo treatment with ip administration of 3-aminobenzamide (10 mg/kg, 1 and 6 hrs after zymosan injection) or nicotinamide (50 mg/kg, 1 and 6 hrs after zymosan injection) significantly decreased mortality, inhibited the development of peritonitis, and reduced peroxynitrite formation. In addition, PARS inhibitors were effective in preventing the development of organ failure because tissue injury and neutrophil infiltration, by myeloperoxidase evaluation, were reduced in the lung, small intestine, and liver. CONCLUSIONS: In conclusion, the major findings of our study are that peroxynitrite and the consequent PARS activation exert a role in the development of multiple organ failure and that PARS inhibition is an effective anti-inflammatory therapeutic tool.

Nitrotyrosine in plasma of celiac disease patients as detected by a new sandwich ELISA.

Inflammation is characterized by increased nitric oxide production. Nitrotyrosine has recently been suggested to be useful as a marker for NO-mediated tissue damage. In context of the development of an ELISA for detection of nitrotyrosine in plasma, monoclonal anti-nitrotyrosine antibodies were developed by injecting mice with nitrated keyhole limpet hemocyanin. The specificity of the antibodies was determined by binding to nitrated BSA, lack of binding to unmodified BSA, tyrosine, 3-chlorotyrosine or phenylalanine and inhibition of binding by nitrotyrosine. The antibodies developed are useful for Western blot analysis and immunohistochemical staining. Using these antibodies a nitrotyrosine sandwich ELISA was developed with a lower detection limit of approximately 0.2 nM. The intra- and interassay variance were 2.4% and 11.9%, respectively. Using this newly developed ELISA, 1.27 +/- 1.03 microM nitrotyrosine was detected in plasma samples of celiac disease patients whereas nitrotyrosine was undetectable in control samples. Elevated nitrotyrosine levels were paralleled by an increase in plasma concentrations of NO-oxidation products (NOx), nitrite and nitrate from 15.1 +/- 6.1 microM in controls to 61.0 +/- 28.2 microM in celiac disease patients. Both nitrotyrosine and NOx levels declined when the patients were on a gluten-free diet, suggesting a relation between intestinal inflammation and plasma nitrotyrosine and NOx levels.

Extensive peroxynitrite activity during progressive stages of central nervous system inflammation.

Nitric oxide (NO) production has been associated with disease activity in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). This free radical can be transformed by superoxide to peroxynitrite, an extremely toxic oxidant which causes lipid peroxidation. In addition, peroxynitrite nitrates tyrosine residues, resulting in nitrotyrosine, which can be identified immunohistochemically. The results of this study indicate that peroxynitrite is formed very early during EAE development, correlating with clinical disease activity. Nitrotyrosine-positive cells display a widespread distribution in brain and spinal cord during severe disease and are associated with both perivascular infiltrates and parenchymal sites. Double-staining procedures demonstrated that a subpopulation of CD11b-positive cells (macrophages/microglia) reacted with nitrotyrosine antibodies. Immunostaining for inducible NO synthase demonstrated a similar distribution as nitrotyrosine staining. These experiments indicate that peroxynitrite is formed during progressive stages of disease activity.

Suppression of IFN-gamma production from Listeria monocytogenes-specific T cells by endogenously produced nitric oxide.

The induction of nitric oxide (NO) by IFN-gamma has been well documented in a variety of experimental settings, but so far there has been no report on whether the endogenously produced NO can suppress IFN-gamma production. In the present study, CD4+ T cells from Listeria monocytogenes-immune mice produced IFN-gamma upon stimulation with specific antigen and NO was generated in culture. When NG-monomethyl-L-arginine (NMMA) was added to the culture at a dose sufficient for the complete blockade of NO production, there was a significant level of enhancement of IFN-gamma production, which was also dose dependently correlated with addition of NMMA. RT-PCR revealed that IFN-gamma mRNA per given amount of total RNA remained the same irrespective of NO blockade by NMMA; however, total RNA recovery was significantly higher in the culture with NMMA. The endogenously produced NO suppressed T-cell proliferation which can be restored by the addition of NMMA. Sodium nitroprusside, a spontaneous NO generator, inhibited T-cell proliferation dose dependently and suppressed IFN-gamma production. Taken together, it may be concluded that NO down-regulates IFN-gamma production mainly by inhibiting T-cell proliferation.

Antimicrobial effect of acidified nitrite on gut pathogens: importance of dietary nitrate in host defense.

Dietary intake of nitrate generates salivary nitrite, which is acidified in the stomach, leading to a number of reactive intermediates of nitrogen, among which are the potentially carcinogenic N-nitrosamines. Acidified nitrite, however, also has antimicrobial activity which coincides with the formation of nitric oxide. The present study examines the antimicrobial effect in vitro of acidified nitrite on Salmonella enteritidis, Salmonella typhimurium, Yersinia enterocolitica, Shigella sonnei, and Escherichia coli O157. First-order regression plots showed a linear inverse relationship of log-transformed proton and nitrite concentrations with MICs and MBCs after 30 min, 2 h, and 24 h of exposure (P < 0.001 for all antibacterial activities). Susceptibility to the acidified nitrate solutions ranked as follows: Y. enterocolitica > S. enteritidis > S. typhimurium = Shigella sonnei > E. coli O157 (P < 0.05). Addition of SCN-, but not that of CI-, increased the antibacterial activity (paired t testing, P < 0.001). Generation of salivary nitrite from dietary nitrate may provide significant protection against gut pathogens in humans.

Aditivos de las presentaciones medicamentosas / Additive of the medical samples

La Tartrazina y Sulfitos son responsables del 90%de las reacciones a los aditivos. Colorantes como Amaranto (Rojo Nro.4), Eritrocina (Rojo Nro.3), Azul Brillante (Azul Nro.1), etc. y conservantes como parabenos, nitratos, benzoatos, etc., son citados por reportes aislados en la literatura como causantes de racciones medicamentosas. En nuestro país la mayoría de prospectos de los medicamentos no detallan los excipientes. Se inició el presente trabajo revisando uno por uno los prospectos adjuntos de las presentaciones farmacéuticas, en farmacias y droguerías de nuestra ciudad. Se agruparon los medicamentos en 5 categorías. Se enviaron cartas a 104 de los Laboratorios, solicitando nos confirmen o nos rectifiquen, si existe error en cuanto a la lista de presentaciones farmacéuticas que adjuntamos. Así tenemos: 1)Medicamentos con sulfitos. Ej: Biletan (comp.), Gentamina (amp.). 2)Medicamentos con tartrazina. Ej: Pankreoflat AD (comp.). 3)Medicamentos que no aclaran excipientes. Ej.: Berco (susp.) 4)Medicamentos que aclaran excipientes, sin sulfitos, ni tartrazina. Ej.: Ventolin (cpto)(jarabe). 5)Medicamentos que no se conocen sus prospectos por falta de existencia. Ej.:Amplidine Balsámico (susp.). Hay laboratorios que colaboran y otros que no contestaron nuestras cartas. El listado de las presentaciones medicamentosas, resultante de este trabajo, permitirá que los médicos alergólogos, tomen conocimiento de los excipientes que en ciertos pacientes sensibles pueden causar reacciones

Ultrastructural and phenotypic changes in Langerhans cells induced in vitro by contact allergens.

Ultrastructural changes of murine Langerhans cells (LC) were examined following exposure of crude epidermal cell suspensions to the contact allergens dinitrochlorobenzene, nickel sulphate and lead nitrate at various concentrations and for various incubation times. An immunogold labelling technique was employed to study changes in surface expression of MHC Class II (Ia) molecules. In all cases, activation of LC was evident after as little as 15 min exposure and was characterized by a marked increase in surface expression of Ia molecules, prominent rough endoplasmic reticulum and numerous ribosomes and lysosomes. Degenerative changes in LC were apparent to varying degrees depending on the allergen, its concentration and the time of incubation. Degenerative changes included swollen mitochondria, membrane disruption or rupture, loss of density of the cytoplasm (cytolysis), loss of dendritic processes and decreased expression of Ia molecules. In the case of dinitrochlorobenzene, degenerative changes were present and usually severe at concentrations greater than 10 micrograms/ml, while exposure to nickel sulphate and lead nitrate was associated with only mild degenerative changes. These observations indicate that contact allergens have a variety of direct effects on LC, including activation and degeneration, which are dose- and time-dependent. Since these alterations of LC were observed in the absence of other immunologically active cells, peripolesis cannot be involved in these events.