Reut Falach

Background Ricin exposure induces a rapid, route-dependent physiological deterioration that significantly limits the window for effective intervention. This study evaluated a wearable photoplethysmography-based monitor (W-PPG-M) for continuous, noninvasive detection of ricin-induced changes in awake swine. Methods Twenty-two female pigs were assigned to control ( n = 5 ), intramuscular exposure ( n = 8 ), or intratracheal exposure ( n = 9 ). A chest-mounted W-PPG-M continuously recorded heart rate, oxygen saturation, cardiac output, stroke volume, systemic vascular resistance, blood pressure parameters, respiratory rate, and pulse pressure. Data were normalized to total monitoring duration and analyzed in 5% time intervals. Results The device consistently identified early compensatory responses across both exposure routes. Increases in heart rate and cardiac output emerged at approximately 40–45% of the monitoring period. In the intramuscular group, this phase was accompanied by a decline in systemic vascular resistance. In the intratracheal group, oxygen saturation diverged from controls at approximately 55% of the monitoring time. The W-PPG-M performance declined during advanced stages of intoxication. Mean arterial pressure remained relatively stable despite invasive evidence of progressive hypotension in the intramuscular group. In addition, the respiratory rate algorithm reached an upper detection limit, failing to capture severe tachypnea observed in the intratracheal group. Conclusions These findings indicate that wearable photoplethysmography monitoring is effective for early detection and trend analysis following ricin exposure but lacks sufficient resolution to characterize late-stage physiological collapse. A hybrid approach combining noninvasive monitoring with targeted invasive measurements may improve translational applicability.

Background: Neutrophils are critical first responders of the innate immune system, rapidly recruited to sites of infection or sterile injury. Upon activation by pathogen- or damage-associated molecular patterns, neutrophils initiate antimicrobial responses, including cytokine release, phagocytosis, and the formation of neutrophil extracellular traps (NETs). While NETosis plays a protective role, excessive NET formation can exacerbate inflammation and tissue damage. Pulmonary exposure to ricin, a potent toxin derived from Ricinus communis, results in acute lung injury characterized by neutrophil infiltration, cytokine production, vascular leakage, and pulmonary edema. This study investigated the contribution of NETosis to ricin-induced lung pathology and explored the therapeutic potential of targeting NETosis with a long acting recombinant DNase I (PRX-119) to attenuate lung injury. Methods: CD1 outbreed mice were pulmonary exposed to ricin, and bronchoalveolar lavage fluid (BALF) and lung tissues were collected at various time points post-exposure. NETosis was assessed by immunofluorescence and Western blot analysis of markers, including peptidyl arginine deiminase 4 (PAD4), citrullinated histone H3 (citH3) and myeloperoxidase (MPO). Therapeutic intervention included administration of a NET-degrading DNase agent in combination with an anti-ricin antibody. Cell-free DNA levels, NETosis markers, neutrophil infiltration, lung histopathology, vascular permeability and the expression of pro- and anti-inflammatory mediators were evaluated. Weight loss and survival were also monitored and compared between anti-ricin monotherapy and combined anti-ricin and plant-produced human recombinant long acting (LA) DNase I (PRX-119), a novel NET degradation therapy. Results: Ricin exposure led to elevated pulmonary levels of PAD4, citH3 and MPO, accompanied by extensive NET formation in both BALF and lung tissue. Mice receiving combined therapy with a newly developed DNase I - based agent (PRX-119) and an anti-ricin antibody treatment exhibited significantly improved survival and reduced weight loss compared to antibody monotherapy. The combined treatment not only significantly reduced NETosis markers, but also improved lung histopathology, reduced vascular leakage and pulmonary edema and altered the levels of proteins involved with pro- or anti-inflammatory effects, Dkk-1, CD93 and Periostin. Conclusions: These findings demonstrate for the first time that NETosis plays a significant pathological role in ricin-induced lung injury. Moreover, they underscore the therapeutic potential of combining advanced NET-degrading agents, specifically PRX-119, an advanced DNAse I under development, with toxin-neutralizing antibodies as a promising strategy to reduce acute lung damage and enhance clinical outcomes.

Ricin is a highly potent toxin that causes severe lung injury upon inhalation by initiating a complex cascade of cellular responses that ultimately leads to cell death. The low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional receptor involved in various physiological processes, including ricin-mediated toxicity. This study explores the role of LRP1 shedding in the development of ricin-induced lung injury. Analysis of bronchoalveolar lavage fluid (BALF) from ricin-intoxicated mice and swine showed a significant increase in soluble LRP1 (sLRP1) levels, whereas serum LRP1 levels remained largely unchanged, suggesting the lungs are the primary source of sLRP1 release. In vitro assays demonstrated the formation of ricin-sLRP1 complexes, indicating that sLRP1 in BALF retained ricin-binding capability. Flow cytometric analysis of lung cells revealed a reduction in both the percentage and total number of LRP1-expressing cells following ricin exposure. Further investigation of specific lung cell populations showed that alveolar epithelial type II (AT-II) cells, despite experiencing significant injury, exhibited minimal LRP1 shedding. No shedding of LRP1 occurred in neutrophils. In contrast, fibroblasts, which were resistant to ricin-induced cell death, exhibited increased shedding of LRP1 and a corresponding decrease in membrane-bound LRP1 expression. This shedding of the LRP1 ectodomain was mediated by metalloproteinases. Immunohistochemical staining further confirmed decreased LRP1 expression in fibroblasts from ricin-exposed mice. Macrophages also showed substantial LRP1 shedding, despite undergoing significant depletion. These findings highlight the complex cell-specific nature of LRP1 shedding in response to ricin intoxication and suggests the potential role of LRP1 in modulation of cellular susceptibility and resistance to ricin-induced lung injury.

Ricin, a highly toxic glycoprotein derived from the seeds of Ricinus communis, poses significant risks in bioterrorism and toxicology due to its rapid absorption and ease of dissemination. Rapid, ultra-sensitive detection is crucial for timely medical intervention and implementing security measures. However, existing methods often lack sufficient sensitivity or require lengthy processing, limiting their utility for trigger-to-treat scenarios. Here, we present an optical modulation biosensing (OMB)-based ricin assay capable of detecting low concentrations of ricin in buffer, plasma, and biological samples. The assay combines magnetic-bead-based target capture with fluorescent signal enhancement, achieving a limit of detection (LoD) of 15 pg/mL in buffer and 62 pg/mL in plasma, with a 4-log dynamic range. Optimized protocols reduced the assay time to 60 min, maintaining an LoD of 114 pg/mL in plasma while preserving accuracy and reproducibility. The assay successfully detected ricin in bronchoalveolar lavage fluid and serum from mice that were intranasally exposed to ricin, with signals persisting up to 48 h post exposure. Its rapid, high-throughput capabilities and simplified workflow make the OMB-based assay a powerful tool for toxicology, forensic analysis, and counter-bioterrorism. This study highlights the OMB platform’s potential as a sensitive and robust diagnostic tool for detecting hazardous biological agents.

Ricin, an extremely potent toxin produced from the seeds of castor plant, Ricinus communis, is ribosome-inactivating protein that blocks cell-protein synthesis. It is considered a biological threat due to worldwide availability of castor beans, massive quantities as a by-product of castor oil production, high stability and ease of production. The consequence of exposure to lethal dose of ricin was extensively described in various animal models. However, it is assumed that in case of aerosolized ricin bioterror attack, the majority of individuals would be exposed to sublethal doses rather than to lethal ones. Therefore, the purpose of current study was to assess short- and long-term effects on physiological parameters and function following sublethal pulmonary exposure. We show that in the short-term, sublethal exposure of mice to ricin resulted in acute lung injury, including interstitial pneumonia, cytokine storm, neutrophil influx, edema and cellular death. This damage was manifested in reduced lung performance and physiological function. Interestingly, although in the long-term, mice recovered from acute lung damage and restored pulmonary and physiological functionality, the reparative process was associated with lasting fibrotic lesions. Therefore, restriction of short-term acute phase of the disease and management of long-term pulmonary fibrosis by medical countermeasures is expected to facilitate the quality of life of exposed survivors.

Ricin, a highly potent plant-derived toxin, is considered a potential bioterrorism weapon due to its pronounced toxicity, high availability, and ease of preparation. Acute damage following pulmonary ricinosis is characterized by local cytokine storm, massive neutrophil infiltration, and edema formation, resulting in respiratory insufficiency and death. A designated equine polyclonal antibody-based (antitoxin) treatment was developed in our laboratory and proved efficacious in alleviating lung injury and increasing survival rates. Although short-term pathogenesis was thoroughly characterized in antitoxin-treated mice, the long-term damage in surviving mice was never determined. In this study, long-term consequences of ricin intoxication were evaluated 30 days post-exposure in mice that survived antitoxin treatment. Significant pulmonary sequelae were demonstrated in surviving antitoxin-treated mice, as reflected by prominent histopathological changes, moderate fibrosis, increased lung hyperpermeability, and decreased lung compliance. The presented data highlight, for the first time to our knowledge, the possibility of long-term damage development in mice that survived lethal-dose pulmonary exposure to ricin due to antitoxin treatment.

Abrin is a highly toxic protein obtained from the seeds of the rosary pea plant Abrus precatorius, and it is closely related to ricin in terms of its structure and chemical properties. Both toxins inhibit ribosomal function, halt protein synthesis and lead to cellular death. The major clinical manifestations following pulmonary exposure to these toxins consist of severe lung inflammation and consequent respiratory insufficiency. Despite the high similarity between abrin and ricin in terms of disease progression, the ability to protect mice against these toxins by postexposure antibody-mediated treatment differs significantly, with a markedly higher level of protection achieved against abrin intoxication. In this study, we conducted an in-depth comparison between the kinetics of in vivo abrin and ricin intoxication in a murine model. The data demonstrated differential binding of abrin and ricin to the parenchymal cells of the lungs. Accordingly, toxin-mediated injury to the nonhematopoietic compartment was shown to be markedly lower in the case of abrin intoxication. Thus, profiling of alveolar epithelial cells demonstrated that although toxin-induced damage was restricted to alveolar epithelial type II cells following abrin intoxication, as previously reported for ricin, it was less pronounced. Furthermore, unlike following ricin intoxication, no direct damage was detected in the lung endothelial cell population following abrin exposure. Reduced impairment of intercellular junction molecules following abrin intoxication was detected as well. In contrast, similar damage to the endothelial surface glycocalyx layer was observed for the two toxins. We assume that the reduced damage to the lung stroma, which maintains a higher level of tissue integrity following pulmonary exposure to abrin compared to ricin, contributes to the high efficiency of the anti-abrin antibody treatment at late time points after exposure.

Longevity of the immune response following viral exposure is an essential aspect of SARS-CoV-2 infection. Mild SARS-CoV-2 infection of K18-hACE2 mice was implemented for evaluating the mounting and longevity of a specific memory immune response. We show that the infection of K18-hACE2 mice induced robust humoral and cellular immunity (systemic and local), which persisted for at least six months. Virus-specific T cells and neutralizing antibody titers decreased over time, yet their levels were sufficient to provide sterile immunity against lethal rechallenge six months post-primary infection. The study substantiates the role of naturally induced immunity against SARS-CoV-2 infection for preventing recurring morbidity.

Severe manifestations of coronavirus disease 2019 (COVID-19) are mostly restricted to distinct groups of people who have preexisting morbidities. Most COVID-19 animal models develop a mild pathology that resolves within a relatively short period of time, reflecting the more prevalent asymptomatic-to-mild performance of the disease observed in humans. Mice are normally unaffected by SARS coronavirus-2 infection, because of the inability of the virus to bind effectively to the murine angiotensin-converting enzyme 2 (ACE2) receptor. We have previously demonstrated that induction of mild and transient pulmonary morbidity, by application of low doses of ricin, rendered CD1 mice to be susceptible to this virus, which was displayed by sustained body weight loss and mortality rates >50%. In the present study, we performed transcriptomic analyses charting the major alterations in gene expression of mice that were pre-exposed to low doses of ricin and then subjected to SARS-CoV-2 infection compared to mice that were solely exposed to ricin or infected with SARS-CoV-2. Mice intoxicated and infected with ricin and SARS-CoV-2 demonstrated a marked stimulation of essential immunity genes and biological pathways involved in the activation of natural-killer response, cell death receptors, cytotoxic T-cells, Toll-like receptor signaling and the NLRP3 inflammasome pathway. At the protein level, an induced early and transient interferon response was recorded which was subsequently suppressed. The activation of this array of genes predicts clinical manifestations that are consistent with severe COVID-19 in humans, thereby establishing the suitability of this unique animal model for the study of severe COVID-19 disease.

Ricin toxin isolated from the castor bean (Ricinus communis) is one of the most potent and lethal molecules known. While the pathophysiology and clinical consequences of ricin poisoning by the parenteral route, i.e., intramuscular penetration, have been described recently in various animal models, the preceding mechanism underlying the clinical manifestations of systemic ricin poisoning has not been completely defined. Here, we show that following intramuscular administration, ricin bound preferentially to the vasculature in both mice and swine, leading to coagulopathy and widespread hemorrhages. Increased levels of circulating VEGF and decreased expression of vascular VE-cadherin caused blood vessel impairment, thereby promoting hyperpermeability in various organs. Elevated levels of soluble heparan sulfate, hyaluronic acid and syndecan-1 were measured in blood samples following ricin intoxication, indicating that the vascular glycocalyx of both mice and swine underwent extensive damage. Finally, by using side-stream dark field intravital microscopy imaging, we determined that ricin poisoning leads to microvasculature malfunctioning, as manifested by aberrant blood flow and a significant decrease in the number of diffused microvessels. These findings, which suggest that glycocalyx shedding and microcirculation dysfunction play a major role in the pathology of systemic ricin poisoning, may serve for the formulation of specifically tailored therapies for treating parenteral ricin intoxication.