Reduced levels of alpha-1-antitrypsin in cerebrospinal fluid of amyotrophic lateral sclerosis patients: a novel approach for a potential treatment.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative motor neuron disease that involves activation of the immune system and inflammatory response in the nervous system. Reduced level of the immuno-modulatory and anti-inflammatory protein alpha-1-antitrypsin (AAT) is associated with inflammation-related pathologies. The objective of the present is to determine AAT levels and IL-23 in the cerebrospinal fluid (CSF) of ALS patients and control group. FINDINGS: CSF samples from newly diagnosed ALS patients and age-matched controls were analyzed for AAT and IL-23 by ELISA and magnetic luminex screening, respectively. A statistically significant reduction of 45 % in mean AAT levels was observed in the CSF of ALS patients (21.4 µg/ml) as compared to the control group (mean 38.8 µg/ml, p = 0.013). A statistically significant increase of 30.8 % in CSF mean levels of the pro-inflammatory cytokine IL-23 was observed in ALS patients (1647 pg/ml) in comparison to the controls (1259 pg/ml, p = 0.012). A negative correlation coefficient (r = -0.543) was obtained by linear regression analysis of the two measured parameters (p = 0.036). CONCLUSIONS: Reduced AAT and elevated IL-23 CSF levels support the notion of neuroinflammatory process occurring in ALS patients. Increasing AAT levels in the patients' nervous system should be further investigated as a new therapeutic approach and a novel potential tool for ALS treatment.

Protective effect of a novel peptide against methylmercury-induced toxicity in rat primary astrocytes.

Methylmercury (MeHg) is an environmental neurotoxicant associated with aberrant central nervous system (CNS) functions. In this study, we examined the protective effect of a novel anti-inflammatory and cytoprotective nonapeptide, termed IIIM1, against MeHg-induced toxicity in cultured rat neonatal primary astrocytes. Astrocytes were pretreated for 66 h with 5 µg/ml IIIM1 (4.95 µM) followed by 6 h exposure to MeHg (5 µM). MeHg significantly increased F(2)-isoprostane generation, a lipid peroxidation biomarker of oxidative injury and this effect was significantly reduced upon pre-treatment with IIIM1. The MeHg-induced increase in levels of prostaglandin E(2) (PGE(2)), biomarkers of inflammatory responses, was also decreased in the peptide-treated cells. Mass spectrometry analysis revealed no chemical or binding interaction between MeHg and IIIM1, indicating that intracellular cytoprotective mechanism of action accounts for the neuroprotection rather than direct intracellular neutralization of the neurotoxicant with the peptide. These findings point to therapeutic potential for IIIM1 in a plethora of conditions associated with reactive oxygen species (ROS) generation. The implication of these findings may prove beneficial in designing new treatment modalities that efficiently suppress neurotoxicity, triggered not only by MeHg, but also by other metals and environmental agents, as well as chronic disease conditions that inherently increase reactive radical production and inflammatory signaling.

Anti-inflammatory effects of peptide fragments of H2A histone and Oryza Sativa Japonica protein.

Anti-inflammatory drugs are often of limited use due to low efficacy and toxic effects. The present study describes the anti-inflammatory effects of a novel nonapeptide termed IIIM1, using the mouse hind paw edema as an experimental model of inflammation. Multiple prophylactic injections of IIIM1 resulted in a significant reduction in carrageenan-induced foot pad swelling, both in mice and rats. A single prophylactic treatment of the peptide caused the maximal effect at 7-9 days between the initial peptide treatment and the subsequent carrageenan injection. A reduced inflammatory reaction was observed in transgenic mice constitutively expressing the peptide. A marked decrease in oxidative burst was observed in activated peritoneal macrophages obtained from peptide-treated mice. Furthermore, the sera of IIIM1-treated mice caused a significant decrease in the oxidative burst of macrophages. In addition, the reduction of hind paw swelling in mice injected with the sera of IIIM1-treated mice strongly suggests the presence of a circulating inducible factor responsible for the anti-inflammatory effect of the peptide. Previous LC/MS/MS analysis revealed the presence of a new peptide, termed RA1, in the sera of IIIM1-treated mice. RA1 was identified as a fragment of the Oryza Sativa Japonica protein. The anti-inflammatory effect of RA1 as evidenced by the reduction in carrageenan-induced hind paw swelling corresponded with the decrease in the oxidative burst of macrophages treated in vitro with this peptide. In conclusion, both IIIM1 and RA1 represent potential agents for the efficient treatment of inflammatory diseases that are currently incurable using presently available drugs.

Peaceful use of disastrous neurotoxicants.

The increasing exposure to environmental neurotoxicants in the last decades caused serious health problems in the world population. Some of the neurotoxic agents are being used in agriculture and household such as insecticides and rodenticides and others are of natural origin like snake and scorpion venoms. Additional group of harmful substances is the chemical warfare agents including nerve and blistering agents that are known for their disastrous effects on neuronal tissues. The present paper presents a combination of epidemiological/clinical and molecular approaches for investigating the effect of certain groups of neurotoxicants on a variety of pathologies. The work of Finkelstein and coworkers describes epidemiological and clinical studies on acute and chronic organophosphate (OP)-induced neurotoxicity in certain populations in Israel. They mainly investigated the neurotoxic effects of low-level long-term exposure to OP in agricultural areas but also dealt with acute exposures as well. A molecular approach to OP mechanism of neuronal injury was described by Milatovic and coworkers. They demonstrated OP-induced oxidative injury in pyramidal neurons in the CA1 hippocampal area and its suppression by antioxidants. Lecht and coworkers described the novel snake venom angioneurins as important mediators of the physiological cross-talk between the cardiovascular and nervous systems. They also showed that under certain conditions these angioneurins may induce pathologies such as tumor development or disruption of the vascular barrier function during envenomation. Additional mechanistic/therapeutic approach was presented by Brodsky, Rosengarten, Proscura, Shapira and Wormser. They developed a novel anti-inflammatory peptide that reduced skin irritation induced by heat and sulfur mustard (SM) stimuli. Since SM causes neuropsychiatric symptoms and alterations in neurological functions this peptide may serve as a potential treatment of neuronal injuries caused by environmental neurotoxicants. These reviews highlight different aspects of neurotoxicity, addressing epidemiology and mechanisms of toxicity; and identifying novel potential therapies.

Amelioration of experimental autoimmune encephalitis by novel peptides: involvement of T regulatory cells.

The purpose of the present study was to develop a peptide for treatment of multiple sclerosis (MS). We have tested the effect of a novel anti-inflammatory peptide (KGHYAERVG, termed IIIM1) on experimental autoimmune encephalitis (EAE), an animal model of MS. Our findings demonstrate significant reduction in neurological score following oral administration of IIIM1. Structural studies revealed that the entire peptide is required for activity. The peptide caused significant reduction in IL17, interferon gamma, IL23 and IL12 production by isolated splenocytes and concomitant elevation of anti-inflammatory cytokines. IIIM1 elevated T regulatory cells (Tregs, CD4(+)CD25(+)FoxP3(+)) in brain and spleen of EAE mice. Similar proliferative effect was observed in isolated human and mouse Tregs in vitro. Stimulation of Tregs by IIIM1 caused production of a new peptide termed RA1 present in Oryza Sativa Japonica group. This Japanese rice peptide ameliorated neurological symptoms in the EAE model. Similar beneficial effect was observed upon oral administration of an extract of Japanese rice. In conclusion, oral treatment with IIIM1 ameliorates EAE symptoms via stimulation of Tregs to proliferate and produce RA1 which reduces EAE symptoms. RA1 might be involved in the relatively low prevalence of MS in Japan and other Japanese rice-eating populations.

From topical antidote against skin irritants to a novel counter-irritating and anti-inflammatory peptide.

The primary purpose of the present study was to investigate the mechanism of the counter-irritating activity of topical iodine against skin lesions induced by chemical and thermal stimuli. The hypothesis that iodine exerts its activity by inducing an endogenous anti-inflammatory factor was confirmed by exposing guinea pig skin to heat stimulus followed by topical iodine treatment and skin extraction. Injection of the extract into naïve guinea pigs reduced heat-induced irritation by 69%. The protective factor, identified as a new nonapeptide (histone H2A 36-44, H-Lys-Gly-Asn-Tyr-Ala-Glu-Arg-Ileu-Ala-OH), caused reduction of 40% in irritation score in heat-exposed guinea pigs. The murine analog (H-Lys-Gly-His-Tyr-Ala-Glu-Arg-Val-Gly-OH, termed IIIM1) reduced sulfur mustard (SM)-induced ear swelling at a dose-dependent bell-shape manner reaching peak activity of 1 mg/kg. Cultured keratinocytes transfected with the peptide were more resistant towards SM than the control cells. The peptide suppressed oxidative burst in activated neutrophils in a concentration-dependent manner. In addition, the peptide reduced glucose oxidase-induced skin edema in mice at a dose-dependent bell-shape manner. Apart from thermal and chemical-induced skin irritation this novel peptide might be of potential use in chronic dermal disorders such as psoriasis and pemphigus as well as non-dermal inflammatory diseases like multiple sclerosis, arthritis and colitis.

Protection from toxicants.

Exposures to skin irritants frequently occur in daily life at the workplace, in laboratories and during housekeeping. Apart from the physical protective countermeasures, there is a need for pharmacological preparations for the topical treatment of the exposed skin to prevent the development of burns. Exposure of the skin to a chemical irritant initiates an inflammatory response which progressively intensifies, leading to epidermal and dermal lesions. Topical treatment with povidone-iodine (PI) or iodine ointment significantly reduced skin damage induced by mustard gas (sulfur mustard), hydrofluoric acid and other chemical irritants. Human studies showed the efficacy of PI and iodine against thermal burns. The combination of anti-inflammatory agents and iodine increased the counterirritating activity. Both human and experimental animal studies demonstrated that the ointment should be immediately applied after occurrence: the earlier the treatment, the better the therapeutic effect. In addition, the ointment should be left on the skin long enough for achieving the therapeutic effect. This simple topical treatment can prevent suffering, skin transplantation and complications associated with skin burns.

Toxic serum factor long after single exposure to organophosphate; a new approach for biomonitoring.

One of the major limitations of current methods of biological detection of exposure to hazardous environmental agents is their inability to detect long-term exposures. In the current study we examined the potential of a new bioassay based on the hypothesis that serum of exposed individuals contains a toxic factor(s) produced by an affected cell/tissue. The procedure included exposure of neuronal PC12 cell cultures to sera of rats treated once with the organophosphate chlorpyrifos. Samples taken 4 weeks after chlorpyrifos exposure reduced nerve growth factor (NGF)-induced neurite outgrowth by 40%. This effect lasted 6 weeks after treatment, whereas motor activity and cholinesterase activity returned to normal levels within 1 week. These results demonstrate the potential of the proposed method to detect environmental exposures long after they have occurred.

Early effects of iodine on DNA synthesis in sulfur mustard-induced skin lesions.

Sulfur mustard (SM) is powerful alkylator and highly cytotoxic blisterogen in both humans and animals. This study in male guinea pigs shows that, at an early stage (5 h) after SM exposure, a marked increase occurred in epithelial nuclear vacuolation, epidermal thickening, and dermal acute inflammation. Topical iodine treatment reduced the severity of these parameters. The rate of DNA synthesis expressed by incorporation of bromodeoxyuridine was reduced upon topical treatment with iodine only or SM only by 46 and 72%, respectively. Iodine treatment following SM exposure exerted an effect similar to that of SM only, indicating that DNA synthesis is not directly involved in the mechanism of action of iodine-induced protection.

Involvement of tumor necrosis factor-alpha in sulfur mustard-induced skin lesion; effect of topical iodine.

Sulfur mustard (SM), also termed mustard gas, is a potent vesicant that elicits an inflammatory response upon exposure of the skin. Evaluation of mouse ear 3 h after SM exposure revealed acute inflammatory-cell aggregates in the vascular beds accompanied by strongly TNF-alpha-positive neutrophils. Eight hours after SM exposure, this phenomenon became intensified and associated with infiltration into the adjacent dermis. In ear skin topically treated with iodine, however, no inflammatory cells were observed 3 h after SM exposure; 8 h postexposure, blood vessels contained very few TNF-alpha-positive inflammatory cells. Since TNF-alpha induction was shown to be associated with reactive oxygen species production, we studied the effect of iodine on activated peritoneal mouse neutrophils. Iodine elicited a concentration-dependent reduction in the oxidative burst of activated neutrophils. Iodine also scavenged hydroxyl radicals generated by glucose oxidase in a concentration-dependent manner. The involvement of TNF-alpha in SM-induced skin toxicity was confirmed by reduction of 49 and 30% in ear edema following administration of 1 and 2 mug anti-TNF-alpha antibodies, respectively. These findings were corroborated by quantitative analysis of the histological findings showing 46% reduction in acute inflammation and no signs of subacute inflammation in the treated group, in contrast to the control group treated with SM only. Other epidermal (microblister formation, ulceration, and necrosis) and dermal (neutrophilia, hemorrhage, and necrosis) parameters also showed marked reductions in the antibodies-treated group in comparison to controls. The combination of iodine and antiTNF-alpha antibodies might constitute a new approach for treatment of SM-exposed individuals.

A chick model for the mechanisms of mustard gas neurobehavioral teratogenicity.

The chemical warfare blistering agent, sulfur mustard (SM), is a powerful mutagen and carcinogen. Due to its similarity to the related chemotherapy agents nitrogen mustard (mechlorethamine), it is expected to act as a developmental neurotoxicant. The present study was designed to establish a chick model for the mechanisms of SM on neurobehavioral teratogenicity, free of confounds related to mammalian maternal effects. Chicken eggs were injected with SM at a dose range of 0.0017-17.0 microg/kg of egg, which is below the threshold for dysmorphology, on incubation days (ID) 2 and 7, and then tests were conducted posthatching. Exposure to SM elicited significant deficits in the intermedial part of the hyperstriatum ventrale (IMHV)-related imprinting behavior. Parallel decreases were found in the level of membrane PKCgamma in the IMHV, while eliciting no net change in cytosolic PKCgamma. The chick, thus, provides a suitable model for the rapid evaluation of SM behavioral teratogenicity and elucidation of the mechanisms underlying behavioral anomalies. The results obtained, using a model that controls for confounding maternal effects, may be replicated in the mammalian model and provide the groundwork for studies designed to offset or reverse the SM-induced neurobehavioral defects in both avian and mammals.

Reduced sulfur mustard-induced skin toxicity in cyclooxygenase-2 knockout and celecoxib-treated mice.

Sulfur mustard (SM), a potent vesicant and chemical warfare agent, induces tissue damage involving an inflammatory response, including vasodilatation, polymorphonuclear infiltration, production of inflammatory mediators, and cyclooxygenase activity. To evaluate the role of cyclooxygenase-1 and -2 (COX-1, COX-2) in sulfur mustard-induced skin toxicity, we applied the agent to the ears of wildtype (WT) and COX-1- and COX-2-deficient mice. In the latter, ear swelling 24 and 48 h after exposure was significantly reduced (P < 0.05) by 55% and 30%, respectively, compared to WT. Quantitative histopathology revealed no epidermal ulceration in COX-2-deficient mice but some degree of severity in WT. COX-2-deficient mice showed significant reductions (P < 0.05) in severity of epidermal necrosis (29%), acute inflammation (42%), and hemorrhage (25%), compared to the WT mice. COX-1 deficiency resulted in significant exacerbation (P < 0.05) in severity of some parameters, including increases of 4.6- and 1.2-fold in epidermal ulceration and epidermal necrosis, respectively, compared to WT. Postexposure treatment of normal male ICR mice with the selective COX-2 inhibitor celecoxib resulted in significant reductions of 27% (P < 0.05) and 28% (P < 0.01) in ear swelling at intervals of 40 and 60 min between exposure and treatment, respectively. Histopathological evaluation revealed significant reductions (P < 0.05) in subepidermal microblister formation (73%) and dermal necrosis (32%), compared to the control group. These findings may indicate that COX-2 participates in the early stages of sulfur mustard-induced acute skin toxicity and that COX-1 might exert some protective function against this chemical insult.

Protective effect of topical iodine containing anti-inflammatory drugs against sulfur mustard-induced skin lesions.

Previous studies have shown the antidotal efficacy of topical iodine at 15 and 30 min post-exposure to sulfur mustard (SM). Here we demonstrate efficacy at longer intervals (20, 30, 45, and 60 min, respectively, for data) using an improved topical povidone-iodine preparation termed N66, which contains steroidal and non-steroidal anti-inflammatory agents. In the mouse, N66 reduced severity of ear edema by 43, 47, 44, and 36%; ear epidermal ulceration by 74, 58, 45, and 58%; and epidermal necrosis by 54, 34, 26, and 31% at the respective time points. A similar effect was observed with encrustation. The healing marker, grade of acanthotic area, showed dramatic increases of 39.6-, 25.3-, 20.9-, and 22-fold. Severity of the dermal parameters, acute inflammation and dermal necrosis, was reduced by 63, 34, 34, and 38% and 80, 54, 54, and 59%, respectively. In guinea pig skin, topical treatment with N66 45 min post-exposure reduced the SM-induced ulceration area by 75%. The histological parameters subepidermal microblister formation, epidermal ulceration, epidermal necrosis, and encrustation were reduced by 63, 61, 41, and 41%, respectively. The healing marker, grade of acanthotic area, was elevated by 73%. N66 induced a statistically significant reduction in two dermal markers for tissue damage: acute inflammation (33%) and dermal necrosis (48%). Reduced skin damage was also observed in areas adjacent the treated sites. The pharmacologically active components of N66 showed additive effect. These findings suggest that the povidone-iodine preparation combined with anti-inflammatory agents functions as a potent antidote against skin lesions induced by SM at relatively long intervals between exposure and treatment.

Protective effect of topical iodine preparations upon heat-induced and hydrofluoric acid-induced skin lesions.

In this study, the protective prophylactic and post-exposure effects of novel topical iodine preparations were demonstrated upon heat- and hydrofluoric acid-induced skin lesions in the haired guinea pig. Prophylactic treatment of thermal bums with a liquid iodine preparation resulted in statistically significant reductions of 39% and 30%, respectively, in acute inflammation and hemorrhage-microscopic dermal parameters indicative of acute tissue damage. A clear trend of iodine-induced reduction in dermal necrosis occurred, and the epidermal healing markers, acanthosis and hyperkeratosis, were increased. Postexposure treatment of thermal burns with an iodine ointment preparation immediately after occurrence also conferred significant therapeutic reduction in parameters of tissue damage such as epidermal ulceration (87%), acute inflammation (58%), and hemorrhage (30%). Gross pathological evaluation showed that prophylactic and postexposure treatments with the liquid iodine preparation significantly reduced the heat-induced ulceration area by 97% and 65%, respectively. In addition, immediate treatment with an ointment iodine formulation significantly decreased the ulceration area by 98%; its tetraglycol vehicle also had a beneficial effect. Postexposure treatment with the iodine ointment proved efficacious upon hydrofluoric acid-induced skin burns. We observed statistically significant reductions of 76% and 68% in ulceration areas at intervals of 5 and 10 minutes between exposure and treatment, whereas a weaker effect was observed at a longer time interval of 15 minutes. Our findings suggest the therapeutic usage of these newly developed iodine preparations for thermally induced and hydrofluoric acid-induced skin burns.

Skin toxicokinetics of mustard gas in the guinea pig: effect of hypochlorite and safety aspects.

Sulfur mustard (SM, mustard gas) is a chemical warfare vesicant that rapidly penetrates the skin due to its hydrophobicity. This study measured the rate of SM disappearance from the skin after topical application of the vesicant. In both fur-covered and hairless animals, the remaining toxicant levels measured 60 min after exposure to undiluted SM were 0.6% and 0.3%, respectively, of the initially applied SM amount. However, SM concentration reached 0.4% of the initial dose 3 h following exposure in female fur-covered guinea pigs. SM quantities extracted from skin of male fur-covered and hairless guinea pigs immediately after 16 min of exposure to SM vapor were 12.2 and 21.8 microg, respectively; levels declined to 1.6 and 1.7 microg at 30 and 15 min following termination of exposure of male fur-covered and hairless guinea pigs, respectively. Three swabbing treatments of undiluted SM-exposed skin with gauze pads soaked in 0.5% hypochlorite caused 68% reduction in skin SM content. Similar findings were obtained when hypochlorite was replaced by water (64% reduction). SM content in the gauze pads was 59, 38 and 25 microg, respectively, for the first, second and third decontamination processes with water. No SM was detected in the gauze pads soaked with hypochlorite. In vitro studies showed that incubation of SM with 0.5% hypochlorite at a ratio of 10:1 (v/v) did not cause SM inactivation, whereas 4% hypochlorite reduced SM levels by 17%. However, at a decontaminant:SM ratio of 1000:1, 0.5% and 4% hypochlorite reduced SM levels by 92% and 99%, respectively. These findings are important for health authorities and regulatory agencies in planning precautionary steps to be taken in case of emergency and in routine laboratory work.

Topical treatment with povidone iodine reduces nitrogen mustard-induced skin collagenolytic activity.

Recently we have shown that post-exposure treatment with povidone iodine (PI) protects against nitrogen and sulfur mustard-induced skin lesions. Since proteolytic activity is involved in skin damage caused by chemical irritants, we have studied the effect of iodine on mechlorethamine (HN2)-induced skin collagenolytic activities in the haired guinea pig model. The matrix metalloproteinase-9 (MMP-9) activity increased by 30, 46, 12 and 23% after 3, 24, 48 and 72 h of HN2 exposure, respectively, whereas the MMP-2 was elevated by 8, 65, 8 and 30%, respectively. Topical treatment with PI at 15 and 120 min after HN2 exposure decreased the MMP-9 activity by 67% and 60%, respectively, when skin was analyzed 3 h after exposure. The same trend was observed in the MMP-2 and MMP-1 activities after PI treatment. A stronger effect of PI treatment 15 min following exposure was observed in skin analyzed 24 h after exposure, i.e. a decrease of 83% and 88% in MMP-9 and MMP-2 activities, respectively. Similar findings were observed with an interval of 120 min between HN2 exposure and PI treatment. A much weaker effect was observed on MMP-1 activity. A similar trend of PI-induced reduction in the three types of collagenase activity was found in skin analyzed 48 and 72 h after exposure. Reduced collagenolytic activity may serve as one of the mechanisms by which iodine protects the skin against chemical insult.