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Abstract Background Immunization with recombinant carboxyl-terminal domain of the heavy chain (Hc domain) of botulinum neurotoxin (BoNT) stimulates protective immunity against native BoNT challenge. Most studies developing a botulism vaccine have focused on the whole Hc; however, since the principal protective epitopes are located within β-trefoil domain (Hcβtre), we hypothesize that immunization with the Hcβtre domain is sufficient to confer protective immunity. In addition, enhancing its uptake subsequent to nasal delivery prompted development of an alternative vaccine strategy, and we hypothesize that the addition of targeting moiety adenovirus 2 fiber protein (Ad2F) may enhance such uptake during vaccination. Results The Hcβtre serotype B immunogen was genetically fused to Ad2F (Hcβtre/B-Ad2F), and its immunogenicity was tested in mice. In combination with the mucosal adjuvant, cholera toxin (CT), enhanced mucosal IgA and serum IgG Ab titers were induced by nasal Hcβtre-Ad2F relative to Hcβtre alone; however, similar Ab titers were obtained upon intramuscular immunization. These BoNT/B-specific Abs induced by nasal immunization were generally supported in large part by Th2 cells, as opposed to Hcβtre-immunized mice that showed more mixed Th1 and Th2 cells. Using a mouse neutralization assay, sera from animals immunized with Hcβtre and Hcβtre-Ad2F protected mice against 2.0 LD50. Conclusion These results demonstrate that Hcβtre-based immunogens are highly immunogenic, especially when genetically fused to Ad2F, and Ad2F can be exploited as a vaccine delivery platform to the mucosa.

Background: The identification of novel therapeutic strategies for ovarian cancer (OC), the most lethal gynecological neoplasm, is of utmost urgency. Here, we have tested the effectiveness of the compound 2c (4-hydroxy-2,6-bis(4-nitrobenzylidene)cyclohexanone 2). 2c interferes with the cysteine-dependent deubiquitinating enzyme (DUB) UCHL5, thus affecting the ubiquitin-proteasome-dependent degradation of proteins., Methods: 2c phenotypic/molecular effects were studied in two OC 2D/3D culture models and in a mouse xenograft model. Furthermore, we propose an in silico model of 2c interaction with DUB-UCHL5. Finally, we have tested the effect of 2c conjugated to several linkers to generate 2c/derivatives usable for improved drug delivery., Results: 2c effectively impairs the OC cell line and primary tumor cell viability in both 2D and 3D conditions. The effectiveness is confirmed in a xenograft mouse model of OC. We show that 2c impairs proteasome activity and triggers apoptosis, most likely by interacting with DUB-UCHL5. We also propose a mechanism for the interaction with DUB-UCHL5 via an in silico evaluation of the enzyme-inhibitor complex. 2c also reduces cell growth by down-regulating the level of the transcription factor E2F1. Eventually, 2c activity is often retained after the conjugation with linkers., Conclusion: Our data strongly support the potential therapeutic value of 2c/derivatives in OC.

This study investigates the viability of developing chitosan-based hydrogels derived from waste shrimp shells for the removal of methylene blue and methyl orange, thereby transforming food waste into advanced materials for environmental remediation. Despite chitosan-based adsorbents being conventionally considered ideal for the removal of negative pollutants, through targeted functionalization with poly(3,4-ethylenedioxythiophene): polystyrene sulfonate (PEDOT:PSS) at varying concentrations, we successfully enhance the hydrogels' efficacy in also adsorbing positively charged adsorbates. Specifically, the incorporation of PEDOT:PSS at a concentration of 10% v / v emerges as a critical factor in facilitating the robust adsorption of dyes. In the case of the anionic dye methyl orange (MO, 10 -5 M), the percentage of removed dye passed from 47% (for beads made of only chitosan) to 66% (for beads made of chitosan-PEDOT:PSS 10%), while, in the case of the cationic dye methylene blue (MB, 10 -5 M), the percentage of removed dye passed from 52 to 100%. At the basis of this enhancement, there is an adsorption mechanism resulting from the interplay between electrostatic forces and π-π interactions. Furthermore, the synthesized functionalized hydrogels exhibit remarkable stability and reusability (at least five consecutive cycles) in the case of MB, paving the way for the development of cost-effective and sustainable adsorbents. This study highlights the potential of repurposing waste materials for environmental benefits, introducing an innovative approach to address the challenges regarding water pollution.

Sjögren's Syndrome (SjS) results in loss of salivary and lacrimal gland excretion due to an autoimmune attack on these secretory glands. Conventional SjS treatments address the symptoms, but not the cause of disease. Recognizing this deficit of treatments to reverse SjS disease, studies were pursued using the fimbriae from enterotoxigenic E. coli, colonization factor antigen I (CFA/I), which has anti-inflammatory properties. To determine if CFA/I fimbriae could attenuate SjS-like disease in C57BL/6.NOD-Aec1Aec2 (SjS) females, the Lactococcus lactis (LL) 301 strain was developed to chromosomally express the cfaI operon. Western blot analysis confirmed CFA/I protein expression, and this was tested in SjS females at different stages of disease. Repeated dosing with LL 301 proved effective in mitigating salivary flow loss and in reducing anti-nuclear antibodies (ANA) and inflammation in the submandibular glands (SMGs) in SjS females and in restoring salivary flow in diseased mice. LL 301 treatment reduced proinflammatory cytokine production with concomitant increases in TGF-β + CD25 + CD4 + T cells. Moreover, LL 301 treatment reduced draining lymph and SMG follicular T helper (Tfh) cell levels and proinflammatory cytokines, IFN-γ, IL-6, IL-17, and IL-21. Such evidence points to the therapeutic capacity of CFA/I protein to suppress SjS disease and to have restorative properties in combating autoimmune disease., (© 2023. The Author(s).)

Purpose: The study's objective is to assess the environmental performance of rice production in Northern Italy, in particular in Piedmont, the first Italian and European district for the rice-growing area, and thus identify the most critical hotspots and agricultural processes. In particular, as a case study, a farm located in Vercelli (VC) has been chosen. Subsequently, the study results were compared with other different cultivation practices to evaluate the most sustainable choice., Methods: The application of the LCA has been performed, highlighting the phases of rice production that have the most significant impact. Then, uncertainty and sensitivity analyses have been made to estimate the robustness of the results and assess the influence of changing some input variables on emission reduction. Finally, multivariate statistical, specifically a principal component analysis (PCA), was conducted to aid the interpretation of the output dataset of this case study. LCA, uncertainty analysis, and sensitivity analysis were performed with SimaPro 9.2.0, using ReCiPe 2016 Midpoint (H) methodology, and PCA with R software., Results and Discussions: The hotspot with the highest environmental load is irrigation, which compared to the other phases impacts more in 15 out of 18 categories, including 12 with impacts greater than + 75%. This is because irrigation causes direct impacts, related to the methanogenesis in rice fields, but also indirect impacts related mainly to the production of the energy mix required to move the large masses of irrigation water. Therefore, different water management systems were compared and results show that the irrigation systems based on intermittent paddy submergence (DSI) could result in - 40% lower impacts, resulting to be the preferable technique over the other irrigation systems analyzed, including the traditional one used in this study., Conclusions: In order to reduce the environmental impacts related to the irrigation process, a water management system characterized by intermittent flooding of the paddy field (DSI) could be used as it reduces the environmental impacts the most (- 40%), while the least suitable system is one characterized by continuous flooding without drought periods, as it causes the highest impacts., Supplementary Information: The online version contains supplementary material available at 10.1007/s11367-022-02109-x., Competing Interests: Conflicts of interestThe authors declare no conflict of interest., (© The Author(s) 2022.)

Colonization factor antigen I (CFA/I) fimbria, an adhesin from enterotoxigenic Escherichia coli, confers protection in murine autoimmune models for type 1 diabetes (T1D), multiple sclerosis, and rheumatoid arthritis. Although CFA/I fimbriae's initial mode of action is in a bystander or in an antigen (Ag)-independent fashion, protection is ultimately dependent upon the induction and/or activation of auto-Ag-specific regulatory T cells (Tregs). However, little is known about how protection transitions from bystander suppression to Ag-specific Tregs. Since dendritic cells (DCs) play an integral role in fate decisions for T cells becoming inflammatory or tolerogenic, the described study tests the hypothesis that Lactococcus lactis expressing CFA/I (LL-CFA/I) stimulates DCs to establish a regulatory microenvironment. To this end, bone marrow-derived dendritic cells (BMDCs) were infected in vitro with LL-CFA/I. Results revealed increased production of IL-10, TGF-β, and indoleamine 2,3-deoxygenase (IDO). Although co-culture of LL-CFA/I infected BMDCs with naïve T cells did not promote Foxp3 expression, TNF-α and IFN-γ production was suppressed. NOD mice orally dosed with LL-CFA/I showed an increase in regulatory plasmacytoid DCs (pDCs) expressing IDO and TGF-β in pancreatic lymph nodes (PaLNs) and spleen three days post-treatment. However, Tregs did not appear in the mucosal inductive sites until much later. These findings show that LL-CFA/I influences specific DC populations to establish tolerance., (Copyright © 2021. Published by Elsevier B.V.)

Background: Sjögren's syndrome (SjS), one of the most common autoimmune diseases, impacts millions of people annually. SjS results from autoimmune attack on exocrine (salivary and lacrimal) glands, and women are nine times more likely to be affected than men. To date, no vaccine or therapeutic exists to treat SjS, and patients must rely on lifelong therapies to alleviate symptoms., Methods: Oral treatment with the adhesin from enterotoxigenic Escherichia coli colonization factor antigen I (CFA/I) fimbriae protects against several autoimmune diseases in an antigen (Ag)-independent manner. Lactococcus lactis, which was recently adapted to express CFA/I fimbriae (LL-CFA/I), effectively suppresses inflammation by the induction of infectious tolerance via Ag-specific regulatory T cells (Tregs), that produce IL-10 and TGF-β. To test the hypothesis that CFA/I fimbriae can offset the development of inflammatory T cells via Treg induction, oral treatments with LL-CFA/I were performed on the spontaneous, genetically defined model for SjS, C57BL/6.NOD-Aec1Aec2 mice to maintain salivary flow., Results: Six-week (wk)-old C57BL/6.NOD-Aec1Aec2 mice were orally dosed with LL-CFA/I and treated every 3 wks; control groups were given L. lactis vector or PBS. LL-CFA/I-treated mice retained salivary flow up to 28 wks of age and showed significantly reduced incidence of inflammatory infiltration into the submandibular and lacrimal glands relative to PBS-treated mice. A significant increase in Foxp3 + and IL-10- and TGF-β-producing Tregs was observed. Moreover, LL-CFA/I significantly reduced the expression of proinflammatory cytokines, IL-6, IL-17, GM-CSF, and IFN-γ. Adoptive transfer of CD4 + T cells from LL-CFA/I-treated, not LL vector-treated mice, restored salivary flow in diseased SjS mice., Conclusion: These data demonstrate that oral LL-CFA/I reduce or halts SjS progression, and these studies will provide the basis for future testing in SjS patients.

Background: The 2020 SARS-CoV-2 pandemic led to community-wide measures affecting parents and children such as school/daycare closures, job losses, and interruptions in medical care for children with chronic diseases. This is the first study to describe the level of stress and mental health of parents of either healthy children or children with chronic conditions, during the 2020 pandemic., Methods: A representative sample of US parents was recruited from 10-17 April 2020. Parents completed online questionnaires about the past 7 days, including the Perceived Stress Scale, Resilient Coping Scale, Self-Efficacy Scale, Kansas Marital Satisfaction Scale, Parental Stress Scale, PROMIS Anxiety and Depression scales and various other pandemic-related stress questions Results: Levels of stressors (e.g., job loss, school closures, etc.) were high during this time (e.g., 79% of children attended home/online school) and parents reported being moderately to highly stressed. Rates of clinical anxiety (44.6%) and depression (42.2%) were high. Parents of children with chronic conditions reported higher levels of stress and worse mental health, but did not differ from other parents in dealing with stress or interruptions in work, child schooling, and marital satisfaction., Discussion: The COVID-19 pandemic has introduced unprecedented levels of stress for parents, especially those of children with chronic conditions. Mental health effects are expected to continue for months/years and preparation is needed to meet an increasing demand for mental health care.

Antigen (Ag)-specific tolerization prevents type 1 diabetes (T1D) in non-obese diabetic (NOD) mice but proved less effective in humans. Several auto-Ags are fundamental to disease development, suggesting T1D etiology is heterogeneous and may limit the effectiveness of Ag-specific therapies to distinct disease endotypes. Colonization factor antigen I (CFA/I) fimbriae from Escherichia coli can inhibit autoimmune diseases in murine models by inducing bystander tolerance. To test if Ag-independent stimulation of regulatory T cells (Tregs) can prevent T1D onset, groups of NOD mice were orally treated with Lactococcus lactis (LL) expressing CFA/I. LL-CFA/I treatment beginning at 6 weeks of age reduced disease incidence by 50% (p < 0.05) and increased splenic Tregs producing both IL-10 and IFN-γ 8-fold (p < 0.005) compared to LL-vehicle treated controls. To further describe the role of these Tregs in preventing T1D, protective phenotypes were examined at different time-points. LL-CFA/I treatment suppressed splenic TNF-α + CD8 + T cells 6-fold at 11 weeks (p < 0.005) and promoted a distinct microbiome. At 17 weeks, IFN-γ + CD4 + T cells were suppressed 10-fold (p < 0.005), and at 30 weeks, pancreatic Tbet + CD4 + T cells were suppressed (p < 0.05). These results show oral delivery of modified commensal organisms, such as LL-CFA/I, may be harnessed to restrict Th1 cell-mediated immunity and protect against T1D.

Purpose of Review: Genetic, socioeconomic and clinical features vary considerably among individuals with type 2 diabetes (T2D) influencing disease development, progression and response to therapy. Although a patient-centred approach to pharmacologic therapy of T2D is widely recommended, patients are often treated similarly, irrespective of the differences that may affect therapeutic response. Addressing the heterogeneity of T2D is a major task of diabetes research to lower the high rate of treatment failure as well as to reduce the risk of long-term complications., Recent Findings: A pathophysiology-based clustering system seems the most promising to help in the stratification of diabetes in terms of complication risk and response to treatment. This urges for clinical studies looking at novel biomarkers related to the different metabolic pathways of T2D and able to inform about the therapeutic cluster of each patient. Here, we review the main settings of diabetes heterogeneity, to what extent it has been already addressed and the current gaps in knowledge towards a personalized therapeutic approach that considers the distinctive features of each patient.

Background: Ovary Carcinoma (OC) is the most lethal gynecological neoplasm due to the late diagnoses and to the common development of resistance to platinum-based chemotherapy. Thus, novel therapeutic approaches are urgently required. In this regard, the strategy of drug repurposing is becoming attractive. By this approach, the effectiveness of a drug originally developed for another indication is tested in a different pathology. The advantage is that data about pharmacokinetic properties and toxicity are already available. Thus, in principle, it is possible to reduce research costs and to speed up drug usage/marketing., Results: Here, some noticeable examples of repurposed drugs for OC, such as amiodarone, ruxolitinib, statins, disulfiram, ormeloxifenem, and Quinacrine, are reported. Amiodarone, an antiarrhythmic agent, has shown promising anti-OC activity, although the systemic toxicity should not be neglected. The JAK inhibitor, Ruxolitinib, may be employed particularly in coadministration with standard OC therapy as it synergistically interacts with platinum-based drugs. Particularly interesting is the use of statin which represent one of the most commonly administered drugs in aged population to treat hypercholesterolemia. Disulfiram, employed in the treatment of chronic alcoholism, has shown anti-OC properties. Ormeloxifene, commonly used for contraception, seems to be promising, especially due to the negligible side effects. Finally, Quinacrine used as an antimicrobial and anti-inflammatory drug, is able to downregulate OC cell growth and promote cell death., Conclusion: Whereas further testing in patients are necessary to better clarify the therapeutic potential of repurposed drugs for OC, it is believed that their use, better if combined with OC targeted delivery systems, can significantly contribute to the development of novel and effective anti-OC treatments., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The unmet need for novel therapeutic options for ovarian cancer (OC) deserves further investigation. Among the different novel drugs, small interfering RNAs (siRNAs) are particularly attractive because of their specificity of action and efficacy, as documented in many experimental setups. However, the fragility of these molecules in the biological environment necessitates the use of delivery materials able to protect them and possibly target them to the cancer cells. Among the different delivery materials, those based on polymers and lipids are considered very interesting because of their biocompatibility and ability to carry/deliver siRNAs. Despite these features, polymers and lipids need to be engineered to optimize their delivery properties for OC. In this review, we concentrated on the description of the therapeutic potential of siRNAs and polymer-/lipid-based delivery systems for OC. After a brief description of OC and siRNA features, we summarized the strategies employed to minimize siRNA delivery problems, the targeting strategies to OC, and the preclinical models available. Finally, we discussed the most interesting works published in the last three years about polymer-/lipid-based materials for siRNA delivery.

The benefits of mucosal vaccines over injected vaccines are difficult to ascertain, since mucosally administered vaccines often induce serum antibody responses of lower magnitude than those induced by injected vaccines. This study aimed to determine if mucosal vaccination using a modified vaccinia virus Ankara expressing human immunodeficiency virus type 1 (HIV-1) gp120 (MVAgp120) prime and a HIV-1 gp120 protein boost could be optimized to induce serum antibody responses similar to those induced by an intramuscularly (i.m.) administered MVAgp120 prime/gp120 boost to allow comparison of an i.m. immunization regimen to a mucosal vaccination regimen for the ability to protect against a low-dose rectal simian-human immunodeficiency virus (SHIV) challenge. A 3-fold higher antigen dose was required for intranasal (i.n.) immunization with gp120 to induce serum anti-gp120 IgG responses not significantly different than those induced by i.m. immunization. gp120 fused to the adenovirus type 2 fiber binding domain (gp120-Ad2F), a mucosal targeting ligand, exhibited enhanced i.n. immunogenicity compared to gp120. MVAgp120 was more immunogenic after i.n. delivery than after gastric or rectal delivery. Using these optimized vaccines, an i.n. MVAgp120 prime/combined i.m. (gp120) and i.n. (gp120-Ad2F) boost regimen (i.n./i.m.-plus-i.n.) induced serum anti-gp120 antibody titers similar to those induced by the intramuscular prime/boost regimen (i.m./i.m.) in rabbits and nonhuman primates. Despite the induction of similar systemic anti-HIV-1 antibody responses, neither the i.m./i.m. nor the i.n./i.m.-plus-i.n. regimen protected against a repeated low-dose rectal SHIV challenge. These results demonstrate that immunization regimens utilizing the i.n. route are able to induce serum antigen-specific antibody responses similar to those induced by systemic immunization. IMPORTANCE Mucosal vaccination is proposed as a method of immunization able to induce protection against mucosal pathogens that is superior to protection provided by parenteral immunization. However, mucosal vaccination often induces serum antigen-specific immune responses of lower magnitude than those induced by parenteral immunization, making the comparison of mucosal and parenteral immunization difficult. We identified vaccine parameters that allowed an immunization regimen consisting of an i.n. prime followed by boosters administered by both i.n. and i.m. routes to induce serum antibody responses similar to those induced by i.m. prime/boost vaccination. Additional studies are needed to determine the potential benefit of mucosal immunization for HIV-1 and other mucosally transmitted pathogens., (Copyright © 2019 American Society for Microbiology.)

IL-35, a relatively newly discovered cytokine belonging to the larger IL-12 family, shows unique anti-inflammatory properties, believed to be associated with dedicated receptors and signaling pathways. IL-35 plays a pivotal role in the development and the function of both regulatory B (Bregs) and T cells (Tregs). In order to further its therapeutic potential, a dairy Lactococcus lactis strain was engineered to express murine IL-35 (LL-IL35), and this recombinant strain was applied to suppress collagen-induced arthritis (CIA). Oral administration of LL-IL35 effectively reduced the incidence and disease severity of CIA. When administered therapeutically, LL-IL35 abruptly halted CIA progression with no increase in disease severity by reducing neutrophil influx into the joints. LL-IL35 treatment reduced IFN-γ and IL-17 3.7- and 8.5-fold, respectively, and increased IL-10 production compared to diseased mice. Foxp3 + and Foxp3 - CD39 + CD4 + T cells were previously shown to be the Tregs responsible for conferring protection against CIA. Inquiry into their induction revealed that both CCR6 + and CCR6 - Foxp3 +or- CD39 + CD4 + T cells act as the source of the IL-10 induced by LL-IL35. Thus, this study demonstrates the feasibility and benefits of engineered probiotics for treating autoimmune diseases.

Pesticides containing lead and arsenic were widely used in the US through the 20th century. Legacy contamination from this use poses a health risk as interest in cultivation of abandoned agricultural lands has grown in recent years. We addressed these risks by quantifying Pb and As in soils and produce from a suburban farm in New Jersey, USA and examining the ability of phosphate-bearing amendments (bone meal, triple super phosphate, manure compost and raised bed soil) in combination with Fe and/or Mn amendments to stabilize these metals and prevent their movement into vegetables. Common produce (tomato, carrot, lettuce, and radish) was grown in soils with 133-307 mg Pb kg -1 and 19-73 mg As kg -1 . Our results suggest that vegetables produced on these soils can have Pb and As at levels above health and safety standards, especially root and leafy green vegetables. Phosphate-bearing amendments can reduce extractable Pb but can increase extractable As in soils, and can have similar effects on vegetables. Iron amendment increased both extractable Pb and As, likely due to the presence of elemental sulfur in the Fe amendment, which lowered soil pH, while Mn amendment had the opposite effect. Most of the Pb and As in vegetables appear to be associated with soil particles adhered to the vegetables, and the contribution from uptake was relatively small except for plots treated with Fe-amendments and for carrots. Thus, proper crop selection, rigorous cleaning, and dust and dirt control are critical to reduce the risk of contaminant exposure through the consumption of garden produce., (Copyright © 2018 Elsevier B.V. All rights reserved.)