Ricin Antibodies' Neutralizing Capacity against Different Ricin Isoforms and Cultivars.

Ricin, a highly toxic protein from Ricinus communis, is considered a potential biowarfare agent. Despite the many data available, no specific treatment has yet been approved. Due to their ability to provide immediate protection, antibodies (Abs) are an approach of choice. However, their high specificity might compromise their capacity to protect against the different ricin isoforms (D and E) found in the different cultivars. In previous work, we have shown the neutralizing potential of different Abs (43RCA-G1 (anti ricin A-chain) and RB34 and RB37 (anti ricin B-chain)) against ricin D. In this study, we evaluated their protective capacity against both ricin isoforms. We show that: (i) RB34 and RB37 recognize exclusively ricin D, whereas 43RCA-G1 recognizes both isoforms, (ii) their neutralizing capacity in vitro varies depending on the cultivar, and (iii) there is a synergistic effect when combining RB34 and 43RCA-G1. This effect is also demonstrated in vivo in a mouse model of intranasal intoxication with ricin D/E (1:1), where approximately 60% and 40% of mice treated 0 and 6 h after intoxication, respectively, are protected. Our results highlight the importance of evaluating the effectiveness of the Abs against different ricin isoforms to identify the treatment with the broadest spectrum neutralizing effect.

Raising antibodies against epoxyscillirosidine, the toxic principle contained in Moraea pallida Bak. (Iridaceae), in rabbits.

Moraea pallida Bak. (yellow tulp) poisoning is the most important plant cardiac glycoside toxicosis in South Africa. The toxic principle, a bufadienolide, is 1α, 2α-epoxyscillirosidine. The aim was to investigate the potential to develop a vaccine against epoxyscillirosidine. Epoxyscillirosidine, proscillaridin and bufalin, were successfully conjugated to hen ovalbumin (OVA), bovine serum albumin (BSA) and keyhole limpet haemocyanin (KLH). There was a low immune response following vaccination of adult male New Zealand White rabbits with epoxyscillirosidine-OVA (n = 3) and OVA (n = 3) using Freund's adjuvant in Trial (T) 1. The immune response improved significantly in T2 following doubling of the dose to 0.8 mg/rabbit and changing the adjuvant to Montanide. In T3, the rabbits (n = 15), allocated into 5 equal groups, vaccinated with proscillaridin-BSA, bufalin-BSA, epoxyscillirosidine-KLH, epoxyscillirosidine-BSA and BSA respectively, using Montanide adjuvant, developed antibodies against the administered immunogens, with epoxyscillirosidine-KLH inducing the highest immune response. Proscillaridin and bufalin antibodies cross-reacted with epoxyscillirosidine in an enzyme linked immunosorbent assay. The conjugation methodology will be adjusted in the future to target optimal conjugation efficiency. Additional vaccination will be conducted in search of neutralizing antibodies against the yellow tulp toxin. The cross-reactivity of proscillaridin and bufalin antibodies with epoxyscillirosidine could be studied in future to explore the potential to prevent yellow tulp poisoning.

Alpha-galactosylceramide (αGalCer) enhances vaccine-induced protection in a model of ricin intoxication.

Alpha-galactosylceramide (αGalCer) is a glycolipid derived from a marine sponge that is a potent activator of both mouse and human invariant natural killer T (iNKT) cells. For that reason, αGalCer is a promising vaccine adjuvant that has been shown to improve both humoral and cellular immunity when co-administered with various vaccines, including candidate vaccines for biodefense. In the current study, we tested the effectiveness of αGalCer as an adjuvant for the clinically-relevant ricin toxin subunit vaccine, RiVax. αGalCer had a potent adjuvant effect, as shown by a rapid onset of anti-ricin IgG titers, accelerated development of serum toxin-neutralizing activity, and enhanced protection from lethal ricin challenge in a mouse model. These results underscore the potential of αGalCer to augment the protective immune response to a vaccine designed to counteract ricin toxin, a fast-acting biothreat agent.

The 9th Conference on Metal Toxicity and Carcinogenesis: The conference overview.

Heavy metals, such as arsenic, chromium, cadmium, nickel, mercury, and uranium are known to cause many human diseases and health complications after occupational or environmental exposure. Consequently, metals are environmental health concerns. This manuscript is an overview of the 9th Conference on Metal Toxicity and Carcinogenesis held in October 2016 in Lexington, Kentucky. Since 2000, this biennial meeting brings together experts in the field to discuss current and prospective research in an effort to advance research pertaining to metal toxicity and carcinogenesis. In this review we summarize the major topics discussed and provide insight regarding current research in the field and an account of the direction in which the field is progressing.

Anti-NMDA (a-NMDAR) receptor encephalitis related to acute consumption of metamphetamine: Relevance of differential diagnosis. / Encefalitis antirreceptores (a-NMDAR) en un paciente con ingesta aguda de metanfetanima: importancia del diagnóstico diferencial.

A 19-year-old male came to the Emergency Room of our hospital due to an episode of dystonic movements and disorientation 4 days after consuming methamphetamine, which evolved to a catatonic frank syndrome and eventually to status epilepticus. Definitive diagnosis was anti-NMDA receptor encephalitis, an acute inflammation of the limbic area of autoimmune origin in which early diagnosis and treatment are key elements for the final outcome. In this case, initial normal tests and previous methamphetamine poisoning delayed diagnosis, because inhaled-methamphetamine poisoning causes similar clinical symptoms to anti-NMDA receptor encephalitis. Methamphetamine poisoning may have caused an immune response in the patient, bringing on the progress of the pathology.

The role of interleukin family in perfluorooctanoic acid (PFOA)-induced immunotoxicity.

Perfluorooctanoic acid (PFOA), a prominent perfluorinated compound (PFC), has been widely detected in natural water bodies worldwide. In this study, zebrafish (Danio rerio) was exposed to nominal concentrations of PFOA (0.05, 0.1, 0.5, and 1 mg/L) for 21 d. After exposure, each fish was decapitated, and the spleen was removed to detect the expression patterns of P65 transcription factor, myeloid differentiation 88, relative interleukins (ILs), and antibody genes. PFOA can stimulate pro-inflammatory cytokine at a low exposure concentration (0.05 mg/L) and can inhibit pro-inflammatory cytokine at higher exposure concentrations (≥ 0.1mg/L). The results of linear correlation analysis indicate that Myd88/NF-κB pathway is one of the important pathways to mediate inflammatory cytokine (IL-1ß and IL-21) in zebrafish spleen. Additionally, the relative mRNA expression level of toll-like receptor 2 (TLR2) at 1mg/L PFOA group was decreased to 56% of its corresponding level in the control. IL secretion disorder is possibly closely related to PFOA-induced TLR2 damage in zebrafish spleen. Furthermore, data show that the trends of PFOA-induced IL secretion have a relationship with Ig-secreting trend. This study demonstrates that PFOA can affect IL expression level through NF-κB, and ILs have an important function in the mediation of Ig secretion.

A recombinant chimeric protein containing B chains of ricin and abrin is an effective vaccine candidate.

Both ricin toxin (RT) and abrin toxin (AT) are 2 important toxin agents as potantial bioweapons. A dual subunit vaccine against RT and AT exposure is a promising option for developing prophylactic vaccination. In this study, we constructed a dual vaccine with RT B chain and AT B chain named RTB-ATB. The RTB-ATB chimeric protein was expressed in Escherichia coli (E. coli), and the purified protein was used to evaluate the immune response by a 2 × 2 × 2 × 2 factorial design. The main effects included dose of RTB-ATB, route of immunization injection, immunization time interval, and dose of native toxins challenge. For 2 × LD(50) challenge of RT or AT, 100% of the RTB-ATB immunized mice survived and regained or exceeded their initial weights within 10 days. For 4 × LD(50) challenge, different routes of immunization injection caused significant difference (P < 0.05), intraperitoneal (i.p.) administration of immunogen protected mice better than the subcutaneous (s.c.) administration. In conclusion, when administered i.p. to mice with 25 µg per mouse and immunization time interval Π in the absence of adjuvant, the chimeric protein elicited a stronger immune response and protected the animals from a dose of native toxins which was 4 times higher than their LD(50) in unvaccinated mice. Besides, the RTB-ATB chimeric protein could induce specific neutralizing antibodies against these 2 toxins. We anticipate that this study will open new possibilities in the preparation of RTB-ATB dual subunit vaccine against the exposure to deadly RT and AT.

Effect of recombinant human IFNγ in the treatment of chronic pulmonary complications due to sulfur mustard intoxication.

Pulmonary problems are among the most common chronic complications of sulfur mustard (SM) intoxication and adversely affect patients' quality-of-life. The present trial investigated the impact of immunotherapy with interferon (IFN)-γ on quality-of-life, respiratory symptoms, and circulating immunologic and oxidative parameters in patients suffering from chronic SM-induced complications. Patients (n = 15) were administered IFNγ (100 µg) every other day for a period of 6 months. Assessment of quality-of-life [using St. George respiratory Questionnaire (SGRQ) and COPD Assessment Test (CAT) indices], the severity and frequency of respiratory symptoms, and serum levels of immunologic [including interleukin (IL)-2, IL-4, IL-6, IL-10, IFNγ, calcitonin gene related peptide (CGRP), matrix metallopeptidase (MMP)-9, and tumor necrosis factor (TNF)-α], oxidative stress [malondialdehyde (MDA) as well as total and reduced glutathione, and catalase and superoxide dismutase (SOD) activity], and fibrogenic [transforming growth factor (TGF)-ß] parameters were performed at baseline and at trial end. The results indicated that IFNγ therapy is associated with improvements in SGRQ (p < 0.001) and CAT (p < 0.001) scores, decreased severity of cough (p = 0.001), dyspnea (p < 0.001), and morning dyspnea (p < 0.001), reduced frequency of sputum production (p < 0.001) and hemoptysis (p < 0.001), and elevated FEV1 (p = 0.065). Serum levels of IL-4 (p < 0.001), IL-6 (p < 0.001), IL-10 (p < 0.001), CGRP (p < 0.001), MMP-9 (p = 0.001), TNFα (p < 0.001), TGFß (p < 0.001) and MDA (p = 0.001) were decreased while those of IL-2 (p < 0.001), IFNγ (p < 0.001), and both total (p = 0.005) and reduced glutathione (p = 0.061) increased by the end of the trial. It was concluded that IFNγ has favorable effects on the quality-of-life and alleviates respiratory symptoms in patients suffering from chronic SM-induced pulmonary complications. A modulation of cytokines and oxidative stress appears responsible for the clinical efficacy of IFNγ.

Alteration of Th1/Th2/Th17 cytokine profile and humoral immune responses associated with chromate exposure.

BACKGROUND: The role of chromate exposure in the deregulation of total lymphocyte and other immune factors is largely unclear. OBJECTIVES: We aimed to examine alteration of the Th1/Th2/Th17 cytokine profile and humoral indicators caused by occupational chromate exposure. METHODS: A cross-sectional study was conducted in two similar workshops (groups 1 and 2) with 106 male occupational workers and 50 matched local controls. Environmental and biological exposures were assessed by measuring chromium concentrations in workplace air, and in whole blood and urine samples of the workers. Cytokines in serum (IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, IL-17A) were determined by CBA assay, while immunoglobin (IgA, IgM, IgG, IgE) and complement (C3, C4) were evaluated by immunonephelometric and ELISA methods. Micronucleus analysis was also used to explore the relationship between genotoxicity and immunotoxicity. RESULTS: Compared with the control group, environmental chromate exposure in groups 1 and 2 was much higher, and the mean values of IL-6, IL-10, IFN-γ, IL-17A and IFN-γ/IL-4 were significantly decreased in group 1. In group 2, IgA and IgG levels were reduced, while C3 and C4 were increased. Levels of IFN-γ, IgG and IgA were all inversely associated with whole blood chromium, while C3 and C4 were positively associated with whole blood chromium (p<0.05). Both IL-10 and IL-17A were inversely associated with urine chromium. Correlations were also found between IL-10, IL-17A and micronucleus (r=-0.329, r=-0.312, respectively). CONCLUSIONS: Occupational exposure to chromate could downregulate the cellular and humoral factors of the immune system.

Protective antibody responses against Clostridium difficile elicited by a DNA vaccine expressing the enzymatic domain of toxin B.

A DNA vaccination approach was used in the current study to screen for the immunogenicity of different fragments of toxin A and toxin B from Clostridium difficile. With this approach, protein antigens do not need to be produced in vitro and the immunogenicity of candidate C. difficile antigens can be identified directly in animals. Codon optimized toxin gene fragments were individually cloned into the DNA vaccine vector and tested in mice and rabbits for their ability to elicit C. difficile toxin-specific antibody responses. Only a subset of the C. difficile toxin fragments, including the C-terminal receptor binding domain of toxin A and a novel N-terminal enzymatic domain of toxin B, were able to elicit protective antibody responses as determined by protection of target cells in a cytotoxicity assay or by preventing death of mice in a passive antibody protection study. Significantly, antibodies elicited by the novel N-terminus of the toxin B DNA vaccine were able to increase the level of protection when used in combination with anti-toxin A antibodies in a toxin challenge model in mice.

[Remaxol in pharmacological correction of long-term disorders caused by acute ethylene glycol poisoning].

The effect of remaxol therapy as a part of the complex acute treatment of ethylene glycol poisoning has been experimentally studied on rats. Special attention was paid to the development of acidosis and hypoxia and a decrease in the functional activity of the urinary and immune systems. It was shown that remaxol is capable of restoring the functional activity of organs and systems susceptible to the toxic effect of ethylene glycol. It is suggested that the therapeutic efficiency of remaxol is based on the normalization of metabolic processes in the cell.

Protective immunity to ricin toxin conferred by antibodies against the toxin's binding subunit (RTB).

The B subunit (RTB) of ricin toxin is a galactose-/N-acetyl galactosamine-specific lectin that promotes attachment and entry of ricin into host cells. RTB is also the archetype of the so-called R-type lectin family, whose members include haemagglutinins of botulinum neurotoxin (BoNT) progenitor toxins, as well as the binding subunits of cytolethal distending toxins. Although RTB is an appealing subunit vaccine candidate, as well as a potential target for immunotherapeutics, the degree to which RTB immunization elicits protective antibodies against ricin toxin remains unresolved. To address this issue, groups of mice were immunized with RTB and then challenged with 5×LD(50)s of ricin administered intraperitoneally. Despite high RTB-specific serum antibody titers, groups of RTB immunized mice were only partially immune to ricin challenge. Analysis of a collection of RTB-specific B cell hybridomas suggested that only a small fraction of antibodies against RTB have demonstrable neutralizing activity. Two RTB-specific neutralizing monoclonal IgG(1) antibodies, 24B11 and SylH3, when passively administered to mice, were sufficient to protect the animals against a 5×LD(50) dose of ricin. Both 24B11 and SylH3 blocked ricin attachment to terminal galactose residues and prevented toxin binding to the surfaces of bone marrow-derived macrophages (BMM), suggesting that they function by steric hindrance and recognize epitopes located on RTB's carbohydrate recognition sub-domains (1α or 2γ). These data raise the possibility of using specific RTB sub-domains, rather than RTB itself, as antigens to more efficiently elicit neutralizing antibodies and protective immunity against ricin.

Neutralising antibodies against ricin toxin.

The Centers for Disease Control and Prevention have listed the potential bioweapon ricin as a Category B Agent. Ricin is a so-called A/B toxin produced by plants and is one of the deadliest molecules known. It is easy to prepare and no curative treatment is available. An immunotherapeutic approach could be of interest to attenuate or neutralise the effects of the toxin. We sought to characterise neutralising monoclonal antibodies against ricin and to develop an effective therapy. For this purpose, mouse monoclonal antibodies (mAbs) were produced against the two chains of ricin toxin (RTA and RTB). Seven mAbs were selected for their capacity to neutralise the cytotoxic effects of ricin in vitro. Three of these, two anti-RTB (RB34 and RB37) and one anti-RTA (RA36), when used in combination improved neutralising capacity in vitro with an IC(50) of 31 ng/ml. Passive administration of association of these three mixed mAbs (4.7 µg) protected mice from intranasal challenges with ricin (5 LD(50)). Among those three antibodies, anti-RTB antibodies protected mice more efficiently than the anti-RTA antibody. The combination of the three antibodies protected mice up to 7.5 hours after ricin challenge. The strong in vivo neutralising capacity of this three mAbs combination makes it potentially useful for immunotherapeutic purposes in the case of ricin poisoning or possibly for prevention.

[Nonspecific resistance in psychotropic agent poisoning complicated with pneumonias].

Nonspecific resistance was studied in patients in acute psychotic poisoning complicated with varying pneumonias. The indices obtained during long (120-min) blood incubation with an object of phagocytosis, namely phagocytic index, phagocytic number, bactericidity index, HCT test, and cytochemical activity index were shown to be of the highest informative value that characterizes the state of nonspecific mechanisms of immune protection. The diminished microphagocytic functional activity along with the severity of pneumonia was ascertained to result in the development of an inflammatory process.

Immunisation with anthrolysin O or a genetic toxoid protects against challenge with the toxin but not against Bacillus anthracis.

Anthrolysin O (ALO) is a toxin produced by Bacillus anthracis, the causative agent of anthrax. It is a member of the cholesterol-dependent cytolysin (CDC) group of toxins, many of which are potential vaccine candidates that protect against their producing organisms. Pore formation by ALO was studied by transmission electron microscopy and pores were found to be consistent with those formed by other members of this toxin family. We constructed and characterised a novel genetic toxoid of anthrolysin O, Delta6mALO, which was able to bind to cells but was incapable of pore-formation or haemolysis. The capacity of the haemolytic and non-haemolytic forms of ALO to protect against challenge with the toxin or B. anthracis was determined. Immunisation with both active and non-haemolytic forms of ALO elicited protection against lethal i.v. challenge with ALO but neither was protective against B. anthracis in a murine i.p. challenge model. Immunisation with another CDC, pneumolysin, did not confer cross-protection against challenge with ALO. Histopathological investigation following lethal i.v. challenge with ALO revealed acute pathology in the lungs with occlusion of alveolar vessels by fibrin deposits.

[Antineutrophil cytoplasmic antibodies in nonvasculitis diseases: clinical correlates and target antigens]. / Anticuerpos anticitoplasma de neutrófilo en enfermedades distintas a las vasculitis idiopáticas: correlaciones clínicas y especificidades antigénicas.

The value of antineutrophil cytoplasmic antibodies (ANCA) in the diagnosis of several types of idiopathic vasculitis has been well-documented: In these diseases the ANCA show two classical immunofluorescence patterns, C-ANCA and P-ANCA, which have antigen specificity for the myeloperoxidase and proteinase 3, respectively. However, the appearance of ANCA in very different diseases other than the mentioned vasculitis, has been documented in recent years. In these diseases, the ANCA generally have atypical immunofluorescence patterns and are directed against neutrophil antigens that are different from the previous two, their clinical value still being under debate.

Anticuerpos anticitoplasma de neutrófilo en enfermedades distintas a las vasculitis idiopáticas: correlaciones clínicas y especificidades antigénicas / Antineutrophil cytoplasmic antibodies in nonvasculitis diseases: clinical correlates and target antigens

Es bien conocido el valor de los anticuerpos anticitoplasma de neutrófilo (ANCA) en el diagnóstico de varios tipos de vasculitis idiopáticas. En estas enfermedades estos autoanticuerpos muestran dos patrones clásicos de inmunofluorescencia, C-ANCA y P-ANCA, que poseen, respectivamente, especificidad antigénica por la proteinasa 3 y la mieloperoxidasa. Sin embargo, en los últimos años se ha documentado la aparición de ANCA en muy diversas patologías distintas a las mencionadas vasculitis. En estas enfermedades los ANCA suelen mostrar patrones atípicos por inmunofluorescencia e ir dirigidos a antígenos neutrofílicos distintos de los dos anteriores, estando su valor clínico aún sujeto a debate (AU)

The value of antineutrophil cytoplasmic antibodies (ANCA) in the diagnosis of several types of idiopathic vasculitis has been well-documented: In these diseases the ANCA show two classical immunofluorescence patterns, C-ANCA and P-ANCA, which have antigen specificity for the myeloperoxidase and proteinase 3, respectively. However, the appearance of ANCA in very different diseases other than the mentioned vasculitis, has been documented in recent years. In these diseases, the ANCA generally have atypical immunofluorescence patterns and are directed against neutrophil antigens that are different from the previous two, their clinical value still being under debate (AU)

Evaluation of different promoter sequences and antigen sorting signals on the immunogenicity of Bacillus subtilis vaccine vehicles.

Recombinant Bacillus subtilis strains, either in the form of spores or vegetative cells, may be employed as safe and low-cost vaccine vehicles. In this study, we studied the role of promoter sequences and antigen-sorting signals on the immunogenicity based on previously constructed B. subtilis episomal expression systems. Mice orally immunized with spores or cells encoding the B subunit of the heat labile toxin (LTB), originally expressed by some enterotoxigenic Escherichia coli (ETEC) strains, under control of the stress-inducible gsiB promoter developed higher anti-LTB serum IgG and fecal IgA responses with regard to vaccine strains transformed with plasmids encoding the antigen under control of IPTG-inducible (Pspac) or constitutive (PlepA) promoters. Moreover, surface expression of the vaccine antigen under the control of the PgsiB promoter enhanced the immunogenicity of vegetative cells, while intracellular accumulation of LTB led to higher antibody responses in mice orally immunized with recombinant B. subtilis spores. Specific anti-LTB antibodies raised in vaccinated mice recognized and neutralized in vitro the native toxin produced by ETEC strains. Nonetheless, only mice orally immunized with recombinant B. subtilis strains, either as vegetative cells or spores, expressing intracellular LTB under the control of the gsiB promoter conferred partial protection to lethal challenges with purified LT. The present report further demonstrates that B. subtilis plasmid-based heterologous protein expression systems are adequate for antigen delivery via the oral route.

Evidence for widespread epithelial damage and coincident production of monocyte chemotactic protein 1 in a murine model of intestinal ricin intoxication.

The development of small-animal models is necessary to understand host responses and immunity to emerging infectious diseases and potential bioterrorism agents. In this report we have characterized a murine model of intestinal ricin intoxication. Ricin administered intragastrically (i.g.) to BALB/c mice at doses ranging from 1 to 10 mg/kg of body weight induced dose-dependent morphological changes in the proximal small intestine (i.e., duodenum), including widespread villus atrophy and epithelial damage. Coincident with epithelial damage was a localized increase in monocyte chemotactic protein 1, a chemokine known to be associated with inflammation of the intestinal mucosa. Immunity to intestinal ricin intoxication was achieved by immunizing mice i.g. with ricin toxoid and correlated with elevated levels of antitoxin mucosal immunoglobulin A (IgA) and serum IgG antibodies. We expect that this model will serve as a valuable tool in identifying the inflammatory pathways and protective immune responses that are elicited in the intestinal mucosa following ricin exposure and will prove useful in the evaluation of antitoxin vaccines and therapeutics.