Circulating Microvesicle-Associated Inducible Nitric Oxide Synthase Is a Novel Therapeutic Target to Treat Sepsis: Current Status and Future Considerations.

To determine whether mitigating the harmful effects of circulating microvesicle-associated inducible nitric oxide (MV-A iNOS) in vivo increases the survival of challenged mice in three different mouse models of sepsis, the ability of anti-MV-A iNOS monoclonal antibodies (mAbs) to rescue challenged mice was assessed using three different mouse models of sepsis. The vivarium of a research laboratory Balb/c mice were challenged with an LD80 dose of either lipopolysaccharide (LPS/endotoxin), TNFα, or MV-A iNOS and then treated at various times after the challenge with saline as control or with an anti-MV-A iNOS mAb as a potential immunotherapeutic to treat sepsis. Each group of mice was checked daily for survivors, and Kaplan-Meier survival curves were constructed. Five different murine anti-MV-A iNOS mAbs from our panel of 24 murine anti-MV-A iNOS mAbs were found to rescue some of the challenged mice. All five murine mAbs were used to genetically engineer humanized anti-MV-A iNOS mAbs by inserting the murine complementarity-determining regions (CDRs) into a human IgG1,kappa scaffold and expressing the humanized mAbs in CHO cells. Three humanized anti-MV-A iNOS mAbs were effective at rescuing mice from sepsis in three different animal models of sepsis. The effectiveness of the treatment was both time- and dose-dependent. Humanized anti-MV-A iNOS rHJ mAb could rescue up to 80% of the challenged animals if administered early and at a high dose. Our conclusions are that MV-A iNOS is a novel therapeutic target to treat sepsis; anti-MV-A iNOS mAbs can mitigate the harmful effects of MV-A iNOS; the neutralizing mAb's efficacy is both time- and dose-dependent; and a specifically targeted immunotherapeutic for MV-A iNOS could potentially save tens of thousands of lives annually and could result in improved antibiotic stewardship.

Inducible Nitric Oxide Synthase in Circulating Microvesicles: Discovery, Evolution, and Evidence as a Novel Biomarker and the Probable Causative Agent for Sepsis.

Background: The sepsis pathology remains an enormous medical problem globally because morbidity and mortality remain unacceptably high in septic patients despite intense research efforts. The economic and societal burden of sepsis makes it the most pressing patient care issue in the United States and worldwide. Sepsis is a dysregulated immune response normally initiated by an infection. The need for an early, accurate, and reliable biomarker test to detect the onset of sepsis and for a targeted sepsis therapy are widely recognized in the biomedical community. Content: This report reviews the published findings relevant to microvesicle-associated inducible nitric oxide synthase (MV-A iNOS) as a novel plasma biomarker for the onset of sepsis including human clinical studies and animal studies. Plasma iNOS as a standalone test and as one of the components of a novel panel of biomarkers to stage the progression of sepsis are presented and discussed in comparison to other biomarkers and other proposed panels of biomarkers for sepsis. Summary: The data strongly support the concept that extracellular plasma MV-A iNOS in circulating microvesicles is centrally involved in the initiation of sepsis, and a diagnostic test based upon plasma iNOS can serve as an early pre-symptomatic warning signal for the onset of sepsis. A novel panel of plasma biomarkers comprised of iNOS, pro-IL-18, pro-IL-33, and Reg-1α is proposed as a multianalyte pre-symptomatic method to stage the onset of sepsis for improved prompt data driven patient care.

Development, characterization, and epitope mapping of a panel of twenty-four monoclonal antibodies specific for human inducible nitric oxide synthase.

A panel of monoclonal antibodies (MAbs) to human inducible nitric oxide synthase (hiNOS) has been developed. By isotype analysis of the MAbs cloned from the 24 different positive hybridomas, 13 were determined to be mouse IgG1, two were mouse IgG2a, two were mouse IgG2b, and the seven others were mouse IgM antibodies: all contained kappa light chains. The anti-hiNOS MAbs were initially characterized by ELISA, RIA, Western blot, and immunocytochemistry, and then they were epitope mapped using synthetic peptides and a three-step mapping procedure. In the first step, each of the 24 MAbs was tested by indirect ELISA for binding to 96 overlapping 18-amino acid-long peptides that span the entire 1153-amino acid length of hiNOS. Eight IgG class anti-hiNOS MAbs were found to bind to one of five different peptides. In the second step, a series of amino terminal and carboxyl terminal truncated peptides were synthesized for each of the five peptides to which one or more of the MAbs bound. Each of the eight anti-hiNOS MAbs was found to bind to the truncated peptides with a unique specificity that identified the amino acid segment involved in binding. The third step in the epitope mapping process utilized three series of overlapping 5-, 6-, 7-, 8-, and 9-amino acid-long peptides for each of these segments and identified the exact amino acids of hiNOS involved in antibody binding. Anti-hiNOS MAbs 2A1-F8, 2D2-B2, 21C10-1D10, and 24B10-2C7 were found to be especially useful in different immunoassays.

Limiting iron availability confers neuroprotection from chronic mild carbon monoxide exposure in the developing auditory system of the rat.

Iron deficiency and chronic mild carbon monoxide (CO) exposure are nutritional and environmental problems that can be experienced simultaneously. We examined the effects of chronic mild CO exposure and iron availability on auditory development in the rat. We propose that chronic mild CO exposure creates an oxidative stress condition that impairs the spiral ganglion neurons. The CO-exposed rat pups had decreased neurofilament proteins and increased copper, zinc-superoxide dismutase (SOD1) in the spiral ganglion neurons. We conclude that the increased amount of SOD1 causes an increase in hydrogen peroxide production that allows the Fenton reaction to occur. This reaction uses both iron and hydrogen peroxide to generate hydroxyl radicals and leads to the development of oxidative stress that impairs neuronal integrity. However, rat pups with decreased iron and CO exposure (ARIDCO) exhibited in their cochlea an up-regulation of transferrin, whereas their expression of neurofilament proteins and SOD1 were similar to control. Consequently, reduced iron availability and the normal expression of SOD1 do not promote oxidative stress in the cochlea. By using basal c-Fos expression as a marker for cellular activation we found a significant reduction in c-Fos expression in the central nucleus of the inferior colliculus in iron-adequate rat pups exposed to CO. By contrast, rather than being reduced, c-Fos expression in the ARIDCO group is the same as for controls. We conclude that the cochlea of rat pups with normal iron availability is selectively affected by mild CO exposure, causing a chronic oxidative stress, whereas limiting iron availability ameliorates the effect caused by mild CO exposure by averting conditions that facilitate oxidative stress.

Mild carbon monoxide exposure and auditory function in the developing rat.

We have examined the influence of chronic mild exposure to carbon monoxide (CO) on cognitive (learning) and auditory function in the developing rat. We have demonstrated that the auditory pathway is compromised at exposures less than 50 ppm, whereas learning was not influenced at 100 ppm. Artificially reared rat pups were exposed to CO during the brain growth spurt and onset of myelination. Spatial learning was assessed using the Morris Water Maze and three tests of auditory function: (1) auditory brainstem conduction times; (2) the amplitude of the eighth nerve's action potential; and (3) otoacoustic emissions carried out on rat pups (age 22- 24 days). The pups were gastrostomy-reared on a rat milk substitute and chronically exposed to CO at discrete concentrations in the range of 12-100 ppm from 6 days of age to post-weaning at 21-23 days of age. We found no difference in auditory brainstem conduction times at all CO concentrations in comparison to non-exposed controls. There was a difference in otoacoustic emissions for test and controls at CO concentrations of 50 ppm but not at lower concentrations. There was a consistent attenuation of the amplitude of the eighth nerve's action potential, even at the lowest CO exposure examined. The attenuation of the amplitude of the action potential of the eighth nerve at 50 ppm carbon monoxide exposure did not completely recover by 73 days of age. We conclude that prolonged mild exposure to carbon monoxide during development causes measurable functional changes at the level of the eighth cranial nerve.

Mild carbon monoxide exposure impairs the developing auditory system of the rat.

The object of this study was to determine if chronic exposure to mild concentrations of CO in air caused changes in the integrity of the inferior colliculus during the most active period of synaptogenesis/auditory development. We examined all subregions of the inferior colliculus (IC) of rats by immunocytochemical approaches after pups were exposed chronically to CO concentrations of, 0, 12.5, 25, and 50 ppm in air starting at Day 8 through 20-22 days of age. Mother-reared pups were compared to the gastrostomy-reared pups with or without CO exposure for basal neural activity, using c-Fos immunoreactivity as a marker. Half the rats were examined at 27 days of age, 5 days after the end of CO exposure, and the other half were examined 50 days later at 75-77 days of age. In the central nucleus of the IC, the number of cells expressing a basal level of c-Fos was decreased significantly in the CO-exposed animals when compared to controls; however, there was little or no difference in the number of cells expressing c-Fos in the other subregions of the IC. We conclude that the central nucleus of the inferior colliculus is affected selectively by mild CO exposure (0.0012% in air) and that this reduction in neuronal activity persists into adulthood.

Mild carbon monoxide exposure diminishes selectively the integrity of the cochlea of the developing rat.

Rat pups were chronically exposed to carbon monoxide (CO) concentrations (12 or 25 ppm) in air starting at day 8, through 22 days of age, to examine the changes in the peripheral auditory system. Gastrostomy-reared rat pups, with or without CO exposure, were used and compared with mother-reared pups. The organ of Corti and the neurons of the spiral ganglion were analyzed for their morphology by using immunochemical and histological techniques. The inner and outer hair cells in the organ of Corti of animals exposed to 12 and 25 ppm CO were not different from the controls. However, at 25 ppm CO exposure, the nerve terminals innervating the inner hair cells were swollen. The somata of neurons in the spiral ganglion showed mild changes in the cytoplasm, and signs of mild vacuolization were observed in myelin covering their central processes. Synaptophysin, a marker for synaptic vesicles, and choline acetyltransferase, a marker for cholinergic terminals, showed no difference in immunoreactivity in CO exposed animals at 12 and at 25 ppm when compared with their age-matched controls. Also, Na(+)K(+) ATPase immunoreactivity patterns were normal compared with controls. Three enzymes were significantly reduced at the 25 ppm CO exposure: Cytochrome oxidase, NADH-TR, and calcium ATPase were decreased in both the organ of Corti and the neurons of the spiral ganglion, and decreased immunostaining for the neurofilament and myelin basic proteins was found. We conclude that components of the cochlea are selectively affected by mild chronic CO exposure during development.