DIBI, a polymeric hydroxypyridinone iron chelator, reduces ocular inflammation in local and systemic endotoxin-induced uveitis.

BACKGROUND/OBJECTIVE: Non-infectious uveitis is an inflammatory disease of the eye commonly treated by corticosteroids, though important side effects may result. A main mediator of inflammation are oxygen free radicals generated in iron-dependent pathways. As such, we investigated the efficacy of a novel iron chelator, DIBI, as an anti-inflammatory agent in local and systemic models of endotoxin induced uveitis (EIU). METHODS: Firstly, the effects of DIBI in systemic EIU in Lewis rats were established. 2 hours post intravenous LPS or LPS/DIBI injections, leukocyte activation and functional capillary density (FCD) were examined using intravital microscopy (IVM) of the iridial microcirculation. Secondly, the toxicity of DIBI was evaluated in BALB/C mice for both acute and chronic dosages through gross ocular examination, intraocular pressure measurements and hematoxylin-eosin staining of ocular tissue. Lastly, three groups of BALB/C mice, control, LPS or DIBI + LPS, were studied to evaluate the effectiveness of DIBI in treating local EIU. Five hours post-local intravitreal (i.v) injection, leukocyte activation and capillary density were examined via IVM. RESULTS: Treatment of systemic EIU with DIBI resulted in a reduction of leukocyte activation and FCD improvement within the iridial microcirculation. Toxicity studies suggested that acute and chronic DIBI administration had no adverse effects in the eye. In the local EIU model, DIBI was shown to reduce leukocyte activation and restored the FCD/DCD ratio, providing evidence for its anti-inflammatory properties. CONCLUSIONS: Our study has provided evidence that DIBI has anti-inflammatory effects in experimental uveitis. Additionally, no local ocular toxicity was observed.

Using mushroom farm and anaerobic digestion wastewaters as supplemental fertilizer sources for growing container nursery stock in a closed system.

Wastewaters from farm and composting operations are often rich in select nutrients that potentially can be reutilized in crop production. Liners of silverleaf dogwood (Cornus alba L. 'Argenteo-marginata'), common ninebark [Physocarpus opulifolius (L.) Maxim.], and Anthony Waterer spirea (Spiraeaxbumalda Burvénich 'Anthony Waterer') were grown in 6L containers filled with a bark-based commercial mix. Plants were fertigated daily via a computer-controlled multi-fertilizer injector with three recirculated fertilizer treatments: (1) a stock (control) solution with complete macro- and micro-nutrients, electrical conductivity (EC) 2.2 dS m(-1); (2) wastewater from a mushroom farm; and (3) process wastewater from anaerobic digestion of municipal solid waste. The wastewaters used in both treatments 2 and 3 were diluted with tap water, and the computer was programmed to amend, dispense and recirculate nutrients based on the same target EC as in treatment 1. For comparison, there was a traditional controlled-release fertilizer treatment [Nutryon 17-5-12 (17N-2P-10K) plus micro-nutrients topdressed at a rate of 39 g/plant, nutrients not recirculated]. All three species responded similarly to the three recirculated fertilizer treatments. Growth with the recirculated treatments was similar and significantly higher than that obtained with controlled-release fertilizer. Throughout the study, the EC measured in wastewater-derived nutrient solutions, and also in the container substrate, were similar or close to those of the control treatment, although there were small to large differences among individual major nutrients. There was no sign of nutrient deficiency or toxicity symptoms to the plants. Small to moderate excesses in concentrations of SO(4), Na, and/or Cl were physiologically tolerable to the species.

Super blue box recycling (SUBBOR) enhanced two-stage anaerobic digestion process for recycling municipal solid waste: laboratory pilot studies.

The super blue box recycling (SUBBOR) process is an enhanced, multi-stage anaerobic digestion process for mixed municipal solid waste (MSW) and other biomass feedstock materials. The technology centers on enhanced high solids, thermophilic digestion after steam-pressure disruption of the ligno-cellulosic fiber components that are recalcitrant to conventional anaerobic digestion. Mixed MSW, rich in organic components but also containing inorganic materials, such as glass, aluminum and steel, as well as non-digestible plastic materials, has been laboratory pilot tested with a fully integrated process train designed to treat and recycle all of the MSW components. Methane yields from the MSW were 0.36 m3 CH4/kg volatile solids (VS) representing a 40% increase over the yield obtained from conventional single stage digestion. The secondary digestion step after steam pressure disruption also provided a 40% improvement in total solids and VS reduction. The residual organic fraction following two-stage digestion was fine in texture and was recovered as a clean peat fraction with reduced contents of heavy metal and other fugitive non-digested contaminants. Mass and energy balance determinations indicated a high degree of MSW diversion from landfill disposal (>80%) was achievable by the SUBBOR process as well as substantial net electrical and thermal energy production. Continuous long-term trials of the SUBBOR process at 25,000 tonnes/year are underway.

Steam pressure disruption of municipal solid waste enhances anaerobic digestion kinetics and biogas yield.

Biomass waste, including municipal solid waste (MSW), contains lignocellulosic-containing fiber components that are not readily available as substrates for anaerobic digestion due to the physical shielding of cellulose imparted by the nondigestible lignin. Consequently, a substantial portion of the potentially available carbon is not converted to methane and the incompletely digested residues from anaerobic digestion generally require additional processing prior to their return to the environment. We investigated and developed steam pressure disruption as a treatment step to render lignocellulosic-rich biomass more digestible and as a means for increasing methane energy recovery. The rapid depressurization after steam heating (240 degrees C, 5 min.) of the nondigested residues following a 30-day primary digestion of MSW caused a visible disruption of fibers and release of soluble organic components. The disrupted material, after reinoculation, provided a rapid burst in methane production at rates double those observed in the initial digestion. This secondary digestion proceeded without a lag phase in gas production, provided approximately 40% additional methane yields, and was accompanied by a approximately 40% increase in volatile solids reduction. The secondary digestate was found to be enriched in lignin and significantly depleted in cellulose and hemi-cellulose components when compared to primary digestate. Thus, steam pressure disruption treatment rendered lignocellulosic substrates readily accessible to anaerobic digestion bacteria and improved both the kinetics of biogas production and the overall methane yield from MSW. Steam pressure disruption is central to a new anaerobic digestion process approach including sequential digestion stages and integrated energy recovery, to improve process yields, provide cogenerated energy for process needs, and to provide effective reuse and recycling of waste biomass materials.

Role of lipid intermediate(s) in the synthesis of serogroup B Neisseria meningitidis capsular polysaccharide.

Neisseria meningitidis serogroup B strain M986 was examined for the involvement of lipid intermediate(s) participating in the biosynthesis of the sialic acid capsular polysaccharide. The addition of exogenous undecaprenyl phosphate, phosphatidylethanolamine, or phosphatidylglycerol to particulate membranes, in the presence of cytidine 5'-monophosphosialic acid, resulted in the stimulation of sialyltransferase activity specifically by undecaprenyl phosphate. Sialyltransferase activity, after delipidation of particulate membrane proteins, was specifically reconstituted by undecaprenyl phosphate. After the addition of 14C-labeled cytidine 5'-monophosphosialic acid to particulate membranes, the level of labeled lipid intermediate(s), extracted by chloroform-methanol (2:1), increased up to a maximum level between 3.75 and 5.0 min, which subsequently decreased to a lower steady-state level. Pulse-chase experiments revealed a transient, solvent-extractable, lipid-linked component. The extracted N-acetylneuraminic acid was in polymeric form. Sequential oxidation and reduction of the extracted radioactivity followed by neuraminidase treatment revealed an average degree of polymerization of four or five N-acetylneuraminic acid residues. Bacitracin-sensitive peptidoglycan was synthesized in vitro by particulate membranes. Cross-competition experiments between peptidoglycan and capsular polysaccharide synthesis by preincubation of precursors of one pathway during synthesis of the other revealed a competitive effect for a common component. This component was believed to be a common pool of undecaprenyl phosphate. A model for the production and regulation of the capsular polysaccharide is proposed.

Influence of nutrient limitation and low pH on serogroup B Neisseria meningitidis capsular polysaccharide levels: correlation with virulence for mice.

Neisseria meningitidis strain M986, which possesses a polyanionic sialic acid capsular polysaccharide, was resistant to the bactericidal effects of normal rabbit serum, but sensitive when immune serum and complement were present. An isogenic strain PRM102, deficient in the ability to produce capsular polysaccharide, was sensitive to normal serum. Strain M986, when grown under conditions of low pH or nutrient limitation, synthesized increased levels of capsular polysaccharide. This was accompanied by an increase in cell surface hydrophilicity and virulence for mice. Cells grown in low-pH, iron-limited medium synthesized the highest concentration of polysaccharide and exhibited the highest cell surface hydrophilicity and virulence among the cases examined. The increase in capsular polysaccharide was partly explained by a decrease in the specific activity of a membrane-bound cytidine monophosphate-N acetylneuraminic acid hydrolase. The results suggest that conditions of nutrient limitation and low pH exert profound effects on the physicochemical nature of the meningococcal cell surface which, in turn, cumulate in enhanced virulence of this organism for mice.

Inflammation triggers hypoferremia and de novo synthesis of serum transferrin and ceruloplasmin in mice.

Oil of turpentine was used to induce an artificial inflammation so that we could study its effect on iron metabolism and on synthesis of serum transferrin and ceruloplasmin in mice. It was found that turpentine-induced inflammation triggered the establishment of a hypoferremic state characterized by low levels of serum iron, followed by recovery and a gradual return to normal plasma iron levels. This turpentine-induced hypoferremia and its subsequent recovery paralleled the hypoferremia obtained during meningococcal infection. Moreover, serum transferrin and ceruloplasmin activity levels increased drastically during the recovery from hypoferremia. [14C]leucine incorporation studies revealed a de novo synthesis of both transferrin and ceruloplasmin. Turpentine-induced hypoferremia was also found to provide a protective effect against meningococcal infection which could be partially reversed by exogenous iron. The results of this study suggest that transferrin and ceruloplasmin may be synthesized partly in response to the altered iron metabolism observed during hypoferremia.

Ceruloplasmin and regulation of transferrin iron during Neisseria meningitidis infection in mice.

The role of ceruloplasmin (ferroxidase I; EC 1.16.3.1) in iron metabolism during experimental Neisseria meningitidis infection was investigated. Plasma ceruloplasmin activity was found to increase greatly in mice during the convalescence phase of iron-controlled infection and after a plasma hypoferremia had occurred. Ceruloplasmin activity-deficient animals became hypoferremic as a result of an impaired release of iron from the reticuloendothelial system as shown by impaired return of reticuloendothelial system-processed heme iron in these mice. Hypoferremia in ceruloplasmin activity-deficient mice was associated with an increased resistance to N. meningitidis infection, an effect reversed readily by ceruloplasmin supplementation or iron addition. This evidence implicated ceruloplasmin activity as an important component in the regulation of the plasma transferrin iron pool and suggested that an important role of additional ceruloplasmin as an acute-phase protein might be related to the requirement of additional transferrin iron. This study also provided further evidence of the importance of transferrin iron and host hypoferremia in bacterial infection.

Mechanism of impaired iron release by the reticuloendothelial system during the hypoferremic phase of experimental Neisseria meningitidis infection in mice.

Hypoferremia, the reduction of plasma transferrin iron levels during infection, has been shown to control Neisseria meningitidis infection in mice. The exact nature of the mechanism that regulates this response has been obscure. We have previously shown that hypoferremia does not result from an accelerated removal of iron from the plasma transferrin pool. In this study, we have examined the processing of iron by the reticuloendothelial system during infection. Normal and hypoferremic meningococcus-infected mice were injected with 59Fe-labeled erythrocytes. Kinetics of uptake and redistribution of the label indicated that during the hypoferremic phase of the infection, reticuloendothelial system-processed iron was not returned to the plasma transferrin pool. Fractionation of hepatic cellular compartments showed that this impaired release of iron resulted from a preferential incorporation of heme-derived iron into the intracellular ferritin pool during the hypoferremic phase of the infection. These findings indicate that this withholding of iron within the intracellular pool leads to hypoferremia and therefore denies the extracellular pathogen its essential iron.

Role of iron in Trypanosoma cruzi infection of mice.

The role of iron in experimental infection of mice with Trypanosoma cruzi was investigated. B6 mice had a transient parasitemia and a transient anemia, both of maximal intensity 28 d after the inoculation of T. cruzi. There was a biphasic hypoferremic host response to infection with T. cruzi with the peak hypoferremia also occurring 28 d after inoculation of the parasite. The mortality rate from infection was increased from 23% in phosphate-buffered saline-treated B6 mice to 50% in a group of B6 mice receiving iron-dextran (P less than or equal to 0.025), whereas depletion of iron stores with the iron chelator desferrioxamine B and an iron-deficient diet provided complete protection of B6 mice (P less than or equal to 0.05). The mortality rate in the highly susceptible C3H strain was reduced from 100% in the control group to 45% (P less than or equal to 0.025) in the iron-depleted group. The tissue iron stores were altered in mice receiving either iron-dextran or desferrioxamine B and an iron-deficient diet. In vitro, T. cruzi was shown to require both a heme and a nonheme iron source for an optimal growth rate. The effects of iron excess or depletion on the outcome of infection with T. cruzi correlated both with the growth requirements of the parasite for iron and with the availability of intracellular iron. Thus, it was suggested that the hypoferremic response, by sequestering iron within intracellular stores, potentially enhanced the pathogenicity of the intracellular parasites. Furthermore, the in vivo effects of iron excess and depletion correlated with an effect of iron on the growth rate and pathogenicity of the parasite.

Physiology of sialic acid capsular polysaccharide synthesis in serogroup B Neisseria meningitidis.

The pathway for biosynthesis of sialic acid capsular polysaccharide was examined in Neisseria meningitidis serogroup B strain M986 and in strain PRM102, an isogenic mutant defective in polysaccharide production. Strain PRM102 was found to possess only 25% of the level of sialyltransferase activity that was found in strain M986, but it had wild-type levels of both the N-acetylneuraminic acid (NANA) condensing enzyme and the CMP-NANA synthetase. A new meningococcal enzyme, a CMP-NANA hydrolase, was found in both meningococcal strains. This enzyme generated CMP and NANA from CMP-NANA, had a Km of 0.88 microM, had a Vmax of 10.75 nmol of NANA produced per h per mg of protein, and was completely inhibited by 45.3 microM CMP. The sialyltransferase, which also had CMP-NANA as substrate, was insensitive to CMP addition. Subcellular fractionation and purification of cytoplasmic and outer membranes on sucrose density gradients revealed that both the sialyltransferase and the CMP-NANA hydrolase were cytoplasmic membrane associated. The NANA condensing enzyme and the CMP-NANA synthetase were found to be cytosolic. A working hypothesis for the regulation of sialic acid polysaccharide synthesis was developed. The CMP-NANA hydrolase with its high affinity for CMP-NANA regulates polysaccharide formation by the sialyltransferase, whereas CMP, a product of both the sialyltransferase and the CMP-NANA hydrolase, modulates the activity of the hydrolase on the cytoplasmic membrane.

Involvement of transferrin and transferrin receptors in human natural killer effector:target interaction.

In this current study one of the determinants of natural killer cell specificity in immunosurveillance against cancer, may be the recognition of transferrin receptors on neoplastic cells by the NK effectors. Human transferrin, when saturated with iron (FeTf), was found to inhibit human natural killer (NK) activity against K562 tumor cells, if included in assay mixtures at physiologically relevant levels. Whereas both FeTf and iron-free transferrin (ApoTf) inhibited initial conjugate formation at the level of the target cell, only FeTf inhibited NK cytolytic activity, as judged by release of chromium from the targets. This suggested a functional role for FeTf on either NK or tumor cells. When either targets or effector lymphocytes were pre-incubated with FeTf, inhibition of killing was strongest when the targets were first exposed to FeTf. The evidence suggested that NK-associated transferrin mediated the interaction with target cells through free target-associated transferrin receptors. The finding that rabbit anti-human transferrin antibody (RaHTf) inhibited killing, when reacted with effector lymphocytes but not with target cells, supported this hypothesis.

Turnover in the transferrin iron pool during the hypoferremic phase of experimental Neisseria meningitidis infection in mice.

Mouse transferrin was used to specifically label the plasma transferrin iron pool for studies of iron kinetics in normal mice and infected mice during the hypoferremic phase of experimental meningococcal infection. The plasma transferrin iron pool of normal mice was found to be very dynamic, with a half-life of iron in the pool of 0.7 h. Iron left the plasma pool, entered the bone marrow, and was released into the blood in erythrocytes. Iron from the transferrin pool also entered the liver and spleen and was presumably in the reticuloendothelial system components of these organs. Most of the iron that had been supplied as transferrin iron was found in erythrocytes by 48 h after injection. Studies with mice infected with Neisseria meningitidis strain M1011 revealed similar kinetics for transferrin iron. There was no redistribution of iron within the various iron pools as a result of infection. Iron turnover in the plasma transferrin pool during the hypoferremic phase was similar to control rates, and iron leaving the pool entered its normal erythroid compartments. The lack of accelerated turnover of plasma iron and the finding that plasma iron was not rerouted to storage compartments during the hypoferremic phase provided good evidence that lactoferrin and leukocytic endogenous mediator were not directly involved in redirecting transferrin iron. Our evidence has implicated an impaired return of reticuloendothelial system-processed iron to the transferrin pool during the hypoferremic response. This appears to be a logical point in the erythroid iron cycle for host-mediated iron sequestration, as the reticuloendothelial system is involved in iron storage and may regulate iron levels in the plasma transferrin pool under normal conditions.

A sensitive and convenient assay procedure for transferrin and its application to the purification of mouse transferrin.

A sensitive procedure employing 55Fe binding was developed for the assay of transferrin. This procedure took advantage of the pH dependence and reversibility of iron binding by transferrin and was applicable to both human and mouse transferrins. Excess iron, after saturation of the transferrin iron-binding sites with 55Fe, supplied as Fe-citrate, was efficiently removed by its binding to Amberlite CG-400 anion-exchange resin. Mouse transferrin was purified from plasma using a combination of ammonium sulphate fractionation and ion-exchange chromatography. Two peaks of pure transferrin were obtained by DEAE-Sepharose chromatography. Mouse transferrin was found to have a molecular weight of 80 000.

Enhancement of Neisseria meningitidis infection in mice by addition of iron bound to transferrin.

Small quantities of iron bound specifically to human transferrin were found to stimulate infection with Neisseria meningitidis strain M1011 in mice. An intraperitoneal injection of 17.5 mg of transferrin carrying 22.7 micrograms of Fe resulted in 100% mortality from infection, as compared with no mortality for the controls which had received saline. Five milligrams of ferri-transferrin (FeTf), carrying 6.5 micrograms of Fe, stimulated and prolonged bacteremia in the mice. Thus, FeTf maintained infection, whereas infection was controlled due to iron limitation in control mice. Comparative studies with apotransferrin (iron-free) revealed that the enhancement of infection was due to the supply of iron. FeTf was also found to relieve an iron limitation of growth achieved by ethylenediaminedihydroxyphenylacetic acid (EDDA) in vitro. FeTf abolished the lag phase for growth of N. meningitidis in a defined medium. The results of this study suggest that human FeTf is an immediate source of iron to N. meningitidis both in vitro and in vivo. These findings support the hypothesis that the levels of iron in the circulating transferrin pool of mice determine the course of experimental N. meningitidis infection.

Increased virulence of Neisseria meningitidis after in vitro iron-limited growth at low pH.

At low pH (6.6) and under conditions of iron limitation, Neisseria meningitidis group B (strain SD1C) exhibited an atypical outer membrane protein profile and an increased relative virulence for the mouse. Cells grown in a buffered medium were effectively deprived of iron by the addition of ethylenediamine-diorthohydroxyphenylacetate. The pH of the medium selected for characteristic colonial morphologies: type M3 predominated at pH 6.6, and type M5 predominated at pH 7.7. A mixed population of M1, M3, and M5 colonies was observed at pH 7.2. Isolated outer membrane proteins were analyzed by sodium dodecyl 99 99 sulfate-polyacrylamide gel electrophoresis, and surface exposed proteins were labeled by the [125I]lactoperoxidase method and subsequently identified by autoradiography. Cells grown at pH 6.6 elaborated a major outer membrane protein (protein III; molecular weight, 69,000), which was also present in the outer membrane of iron-limited cells grown at pH 7.2. At pH 7.2 in an iron-sufficient medium, protein III was present only in small quantities in sodium dodecyl sulfate-polyacrylamide gel was present only in small quantities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. A study of the relative virulence (50% lethal dose) of the meningococcus for C57/BL mice revealed that iron-limited cells grown at low pH had an increased relative virulence 1,200-fold (50% lethal dose, 4.0 CFU) greater than that of cells grown in the same medium but at pH 7.2 and with sufficient iron. These studies indicate that pH and iron can be important factors in the determination of meningococcal virulence.

Differences in virulence for mice between disease and carrier strains of Neisseria meningitidis.

The virulence of 11 prototype strains of Neisseria meningitidis, which had been used in the development of the serotyping scheme for serogroup B meningococci, were examined in mice treated with iron dextran. These strains, together with those previously examined, allowed for a good assessment of the virulence differences between carrier and disease strains. All of a total of 17 disease strains displayed virulence for mice (60% with high virulence), whereas only approximately 50% of 13 carrier strains possessed virulence (only 15% with high virulence). Because the ability to initiate infection in mice is independent of exogenous iron, this model system for infection appears particularly suited to studies of the physiological bases for virulence in N. meningitidis.

Iron-controlled infection with Neisseria meningitidis in mice.

An iron-controlled infection was obtained after the intraperitoneal infection of Neisseria meningitidis strain M1011 into normal mice. The infection progressed rapidly but then disappeared in concert with the disappearance of plasma transferrin iron. Parenteral iron dextran enhanced and prolonged the infection in mice at dosages above 15 mg of Fe per kg. Studies on the distribution of iron dextran within the physiological iron pools and the importance of timing with the iron dextran addition indicated that high serum iron, available early during infection, was necessary to promote infection. High levels of iron in the reticuloendothelial system did not stimulate infection. A working hypothesis to explain the roles of iron in infection was developed: N. meningitidis obtains iron for growth from the transferrin pool, and iron dextran maintains transferrin iron levels during infection.

Neisseria meningitidis infection in mice: influence of iron, variations in virulence among strains, and pathology.

The influence of iron on Neisseria meningitidis infection in C-57 mice was examined. Iron sulfate, ferric ammonium citrate, and iron sorbitol citrate all proved to be too toxic for use as infection-enhancing agents. Iron dextran displayed an extremely low toxicity, enhanced infection in a dose-dependent manner, and resulted in infection enhancement factors approaching 10(9) for virulent strains of N. meningitidis. Fatal iron dextran-enhanced infection was shown to be reversible by in vivo chelation of iron. Virulent strains of N. meningitidis produced symptoms of infection and pathological lesions in mice both with and without iron pretreatment, but an avirulent strain failed to produce symptoms of infection or pathological lesions, regardless of iron administration. Iron dextran-enhanced infection in mice proved to be a useful model for the examination of virulence of various N. meningitidis strains. All of 9 isolates from clinical disease possessed virulence, whereas only 3 of 10 isolates from carriers possessed virulence, when examined by using this model.

Trypsin-uncoupled synthesis and secretion of yeast invertase: implications for the mechanism of secretion.

The subcellular distribution of invertase was examined after synthesis and secretion by sphaeroplasts had been uncoupled by the addition of 30 microgram mL-1 trypsin. Sphaeroplasts secreted only the high molecular weight invertase during uncoupling by trypsin. The level of low molecular weight, 'small' invertase in the soluble internal pool was found to be elevated by over fivefold, and the membrane-associated pool was found to contain low molecular weight invertase in addition to intermediate molecular weight invertase, after 1.5 h of trypsin treatment. Purified plasma membranes from trypsin-treated sphaeroplasts had no detectable mannan synthetase activity. On the basis of these and previous findings, a working hypothesis wherein invertase is synthesized on the internal surface of the plasma membrane and glycosylated during its transit to the external surface is presented.