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Introduction: In causal inference, the correct formulation of the scientific question of interest is a crucial step. The purpose of this study was to apply causal inference principles to external control analysis using observational data and illustrate the process to define the estimand attributes.Methods: This study compared long-term survival outcomes of a pooled set of three previously reported randomized phase 3 trials studying patients with metastatic non-small cell lung cancer receiving front-line chemotherapy and similar patients treated with front-line chemotherapy as part of routine clinical care. Causal inference frameworks were applied to define the estimand aligned with the research question and select the estimator to estimate the estimand of interest.Results: The estimand attributes of the ideal trial were defined using the estimand framework. The target trial framework was used to address specific issues in defining the estimand attributes using observational data from a nationwide electronic health record-derived de-identified database. The two frameworks combined allow to clearly define the estimand and the aligned estimator while accounting for key baseline confounders, index date, and receipt of subsequent therapies. The hazard ratio estimate (point estimate with 95% confidence interval) comparing the randomized clinical trial pooled control arm with the external control was close to 1, which is indicative of similar survival between the two arms.Discussion: The proposed combined framework provides clarity on the causal contrast of interest and the estimator to adopt, and thus facilitates design and interpretation of the analyses.

The most successful alternative to traditional surgery for ocular muscle spasm treatment is the intramuscular injection of botulinum toxin (BTX), which allows the maintenance of the muscle dynamics and the absence of scars. However, the main BTX disadvantage is its nonpermanent effect. A possible way for overcoming this obstacle could be represented by the enzymatic surgery using plant toxins known as ribosome-inactivating proteins (RIPs). In this paper, two highly toxic RIPs, namely, ricin and stenodactylin, were considered in a preliminary study for their possible use in the treatment of strabismus and oculofacial dystonias, as alternatives to BTX. Both RIPs showed a strong cytotoxic effect against rhabdomyosarcoma cell lines and myotube differentiated cells, with stenodactylin being about 10-fold more toxic than ricin. Moreover, stenodactylin showed a much higher cytotoxicity on myoblasts than on rhabdomyosarcoma cells. In our experimental conditions, stenodactylin did not damage conjunctival cells. Despite the limitations due to in vitro experiments, our data show that the high cytotoxicity of stenodactylin allows the use of a very low dose and, consequently, of very low injection volumes. This can represent a great advantage in the case of in vivo locoregional treatment. Furthermore, it is possible to modulate the chemoablation of myocytes while destroying myoblasts, thus reducing regenerative phenomena. The risk of cytotoxicity to surrounding tissues would be strongly reduced by the low injected volume and the relative resistance of conjunctival cells. In conclusion, our data suggest that stenodactylin and ricin could represent potential candidates to substitute BTX in ocular dystonia therapy.

Following the invitation of Biomedicines, we decided to accept the project of this Special Issue because we believe that in many situations gender prejudices still exist and put women in a disadvantaged position for the dissemination of their research, preventing the scientific community from benefiting from a plurality of voices in the interpretation of scientific research [...]

Taking into account the patient’s gender is the first step towards more precise and egalitarian medicine. The gender-related divergences observed in purine catabolism and their pathological consequences are good examples of gender medicine differences. Uric acid is produced by the activity of xanthine oxidoreductase (XOR). The serum levels of both XOR activity and uric acid differ physiologically between the genders, being higher in men than in women. Their higher levels have been associated with gout and hypertension, as well as with vascular, cardiac, renal, and metabolic diseases. The present review analyzes the gender-related differences in these pathological conditions in relation to increases in the serum levels of XOR and/or uric acid and the opportunity for gender-driven pharmacological treatment.

Introduction: Alectinib was found to have superior efficacy to crizotinib in the phase 3 ALEX study and is a preferred initial treatment for patients with advanced ALK-positive NSCLC. To understand the efficacy of alectinib in U.S. clinical practice, we conducted a retrospective real-world comparative effectiveness analysis of first-line alectinib versus crizotinib. Methods: Adults with advanced ALK-positive NSCLC who received first-line alectinib (from December 11, 2015) or crizotinib (from January 1, 2014) were included from a real-world database. Propensity scores were applied to balance baseline characteristics. Real-world data (RWD), including real-world progression-free survival (rwPFS), real-world overall survival, real-world time to new central nervous system (CNS) metastases, and outcomes in patients with or without baseline CNS metastases were analyzed. The ALEX-like RWD cohort (filtered by ALEX laboratory eligibility criteria) was used to compare real-world comparative effectiveness with ALEX. Results: The RWD cohort comprised 364 patients (141 alectinib; 223 crizotinib); rwPFS (weighted hazard ratio [wHR] = 0.46, 95% confidence interval [CI]: 0.33–0.65) and real-world overall survival (wHR = 0.46, 95% CI: 0.31–0.69) were significantly improved with alectinib versus crizotinib. In patients with baseline brain scans, a substantial rwPFS benefit was found regardless of baseline CNS metastases. Real-world time to new CNS metastases was delayed with alectinib versus crizotinib in patients with (wHR = 0.28, 95% CI: 0.16–0.52) and without (wHR = 0.42, 95% CI: 0.24–0.76) baseline CNS metastases. The ALEX-like RWD cohort comprised 325 patients (120 alectinib; 205 crizotinib); alectinib was found to have similar rwPFS benefits with ALEX. Conclusions: Outcomes were significantly improved with first-line alectinib versus crizotinib in patients with advanced ALK-positive NSCLC in the U.S. real-world setting.

Human xanthine oxidoreductase (XOR) is a multilevel regulated enzyme, which has many physiological functions, but which is also involved in several pathological processes. The contributions of Stirpe and his research group at the University of Bologna, to the development of knowledge on the XOR enzyme and its implications in physiological and pathological processes made a breakthrough in this field. Furthermore, their pioneering results paved the way for many research lines that are still actual and that have relevant clinical implications.

Ribosome-inactivating proteins (RIPs) are plant toxins that were identified for their ability to irreversibly damage ribosomes, thereby causing arrest of protein synthesis and induction of cell death. The RIPs purified from Adenia plants are the most potent ones. Here, we describe a novel toxic lectin from Adenia heterophylla caudex, which has been named heterophyllin. Heterophyllin shows the enzymatic and lectin properties of type 2 RIPs. Interestingly, in immunoreactivity experiments, heterophyllin poorly cross-reacts with sera against all other tested RIPs. The cytotoxic effects and death pathways triggered by heterophyllin were investigated in three human-derived cell lines: NB100, T24, and MCF7, and compared to ricin, the most known and studied type 2 RIP. Heterophyllin was able to completely abolish cell viability at nM concentration. A strong induction of apoptosis, but not necrosis, and the involvement of oxidative stress and necroptosis were observed in all the tested cell lines. Therefore, the enzymatic, immunological, and biological activities of heterophyllin make it an interesting molecule, worthy of further in-depth analysis to verify its possible pharmacological application.

Immune checkpoint mechanisms are important molecular cell systems that maintain tolerance toward autoantigens in order to prevent immunity-mediated accidental damage. It is well known that cancer cells may exploit these molecular and cellular mechanisms to escape recognition and elimination by immune cells. Programmed cell death protein-1 (PD-1) and its natural ligand programmed cell death ligand-1 (PD-L1) form the PD-L1/PD-1 axis, a well-known immune checkpoint mechanism, which is considered an interesting target in cancer immunotherapy. In fact, the expression of PD-L1 was found in various solid malignancies and the overactivation of PD-L1/PD-1 axis results in a poor patient survival rate. Breaking PD-L1/PD-1 axis, by blocking either the cancer side or the immune side of the axis, is currently used as anti-cancer strategy to re-establish a tumor-specific immune response. For this purpose, several blocking antibodies are now available. To date, three anti-PD-L1 antibodies have been approved by the FDA, namely atezolizumab, durvalumab and avelumab. The main advantages of anti-PD-L1 antibodies arise from the overexpression of PD-L1 antigen by a high number of tumor cells, also deriving from different tissues; this makes anti-PD-L1 antibodies potential pan-specific anti-cancer molecules. Despite the good results reported in clinical trials with anti-PD-L1 antibodies, there is a significant number of patients that do not respond to the therapy. In fact, it should be considered that, in some neoplastic patients, reduced or absent infiltration of cytotoxic T cells and natural killer cells in the tumor microenvironment or presence of other immunosuppressive molecules make immunotherapy with anti-PD-L1 blocking antibodies less effective. A strategy to improve the efficacy of antibodies is to use them as carriers for toxic payloads (toxins, drugs, enzymes, radionuclides, etc.) to form immunoconjugates. Several immunoconjugates have been already approved by FDA for treatment of malignancies. In this review, we focused on PD-L1 targeting antibodies utilized as carrier to construct immunoconjugates for the potential elimination of neoplastic cells, expressing PD-L1. A complete examination of the literature regarding anti-PD-L1 immunoconjugates is here reported, describing the results obtained in vitro and in vivo. The real potential of anti-PD-L1 antibodies as carriers for toxic payload delivery is considered and extensively discussed.

Saporin is a type 1 ribosome-inactivating protein widely used as toxic payload in the construction of targeted toxins, chimeric molecules formed by a toxic portion linked to a carrier moiety. Among the most used carriers, there are large molecules (mainly antibodies) and small molecules (such as neurotransmitters, growth factors and peptides). Some saporin-containing targeted toxins have been used for the experimental treatment of several diseases, giving very promising results. In this context, one of the reasons for the successful use of saporin lies in its resistance to proteolytic enzymes and to conjugation procedures. In this paper, we evaluated the influence of derivatization on saporin using three heterobifunctional reagents, namely 2-iminothiolane (2-IT), N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and 4-succinimidyloxycarbonyl-α-methyl-α-[2-pyridyldithio]toluene (SMPT). In order to obtain the highest number of inserted -SH groups with the lowest reduction of saporin biological activities, we assessed the residual ability of saporin to inhibit protein synthesis, to depurinate DNA and to induce cytotoxicity after derivatization. Our results demonstrate that saporin maintains an excellent resistance to derivatization processes, especially with SPDP, and permit us to define reaction conditions, in which saporin biological properties may not be altered. Therefore, these findings provide useful information for the construction of saporin-based targeted toxins, especially with small carriers.

Cardiovascular diseases (CVD) are the leading cause of global mortality and their pathogenesis lies mainly in the atherosclerotic process. There are close connections linking oxidative stress and inflammation to endothelial dysfunction, atherosclerosis and, consequently, to CVD. This review focuses on the role of xanthine oxidoreductase (XOR) and its products on the development of chronic inflammation and oxidative stress, responsible for atheromatous plaque formation. Evidence is reported that an excessive level of XOR products favors inflammatory response and plaque development, thereby promoting major cardiovascular risk factors. Also, the relationship between hyperuricemia and hypertension as well as between XOR activity and CVD is confirmed. In spite of the increasing number of clinical studies investigating the output of cardiovascular patients treated with urate-lowering therapies (including uricosuric drugs, XOR inhibitors and recombinant uricase) the results are still uncertain. The inhibition of XOR activity appears more promising than just the control of uricemia level in preventing cardiovascular events, possibly because it also reduces the intracellular accumulation of urate, as well as the production of reactive oxygen species. However, XOR inhibition also reduces the availability of the multifaced mediator nitric oxide and, at present, can be recommended only in hyperuricemic patients.

Human xanthine oxidoreductase (XOR) is a multiple-level regulated enzyme, resulting from a complicated evolutionary process that assigned it many physiological roles. The main XOR activities are: (i) xanthine dehydrogenase (XDH) activity that performs the last two steps of purine catabolism, from hypoxanthine to uric acid; (ii) xanthine oxidase (XO) activity that, besides purine catabolism, produces reactive oxygen species (ROS); (iii) nitrite reductase activity that generates nitric oxide, contributing to vasodilation and regulation of blood pressure; (iv) NADH oxidase activity that produces ROS. All these XOR activities contribute also to metabolize various endogenous and exogenous compounds, including some drugs. About XOR products, it should be considered that (i) uric acid is not only a proinflammatory agent, but also a fundamental antioxidant molecule in serum and (ii) XOR-derived ROS are essential to the inflammatory defensive response. Although XOR has been the object of a large number of studies, most of them were focused on the pathological consequences of its activity and there is not a clear and schematic picture of XOR physiological roles. In this review, we try to fill this gap, reporting and graphically schematizing the main roles of XOR and its products.

Oxidative stress plays an essential role in the pathogenesis of chronic diseases such as cardiovascular diseases, diabetes, neurodegenerative diseases, and cancer. Long term exposure to increased levels of pro-oxidant factors can cause structural defects at a mitochondrial DNA level, as well as functional alteration of several enzymes and cellular structures leading to aberrations in gene expression. The modern lifestyle associated with processed food, exposure to a wide range of chemicals and lack of exercise plays an important role in oxidative stress induction. However, the use of medicinal plants with antioxidant properties has been exploited for their ability to treat or prevent several human pathologies in which oxidative stress seems to be one of the causes. In this review we discuss the diseases in which oxidative stress is one of the triggers and the plant-derived antioxidant compounds with their mechanisms of antioxidant defenses that can help in the prevention of these diseases. Finally, both the beneficial and detrimental effects of antioxidant molecules that are used to reduce oxidative stress in several human conditions are discussed.

Stenodactylin, a highly toxic type 2 ribosome-inactivating protein purified from the caudex of Adenia stenodactyla Harms, is a potential anticancer drug candidate. Previous studies demonstrated that stenodactylin induces apoptosis and necroptosis in treated cells, involving the production of reactive oxygen species. We analyzed the effect of stenodactylin on Raji and Ramos (Human Burkitt’s lymphoma cells) and MOLM-13 (acute myeloid leukemia cells). Moreover, we focused on the early events in MOLM-13 cells that characterize the cellular response to the toxin by whole-genome microarray analysis of gene expression. Treatment with stenodactylin induced the depurination of 28S rRNA within 4 h and increased the phosphorylation of p38 and JNK. A time-dependent activation of caspase 1, 2, 8, 9, 3/7 was also observed. Genome-wide gene expression microarray analysis revealed early changes in the expression of genes involved in the regulation of cell death, inflammation and stress response. After 4 h, a significant increase of transcript level was detectable for ATF3, BTG2, DUSP1, EGR1, and JUN. Increased upstream JUN signaling was also confirmed at protein level. The early response to stenodactylin treatment involves inflammatory and apoptotic signaling compatible with the activation of multiple cell death pathways. Because of the above described properties toward acute myeloid leukemia cells, stenodactylin may be a promising candidate for the design of new immunoconjugates for experimental cancer treatment.

The use of radio- and chemotherapeutic agents in cancer therapy have demonstrated evident antitumor effects, but also limitations (remarkable side-effects due to lack of selectivity for tumor cells, development of drug resistance, and occurrence of secondary malignancies) [...]

The concept of “magic bullets”, i [...]

Kirkiin is a new type 2 ribosome-inactivating protein (RIP) purified from the caudex of Adenia kirkii with a cytotoxicity compared to that of stenodactylin. The high toxicity of RIPs from Adenia genus plants makes them interesting tools for biotechnology and therapeutic applications, particularly in cancer therapy. The complete amino acid sequence and 3D structure prediction of kirkiin are here reported. Gene sequence analysis revealed that kirkiin is encoded by a 1572 bp open reading frame, corresponding to 524 amino acid residues, without introns. The amino acid sequence analysis showed a high degree of identity with other Adenia RIPs. The 3D structure of kirkiin preserves the overall folding of type 2 RIPs. The key amino acids of the active site, described for ricin and other RIPs, are also conserved in the kirkiin A chain. Sugar affinity studies and docking experiments revealed that both the 1α and 2γ sites of the kirkiin B chain exhibit binding activity toward lactose and D-galactose, being lower than ricin. The replacement of His246 in the kirkiin 2γ site instead of Tyr248 in ricin causes a different structure arrangement that could explain the lower sugar affinity of kirkiin with respect to ricin.

Sarcomas are one of the most difficult type of cancer to manage and treat because of their extremely heterogeneous molecular and morphological features. Despite the progress made over the years in the establishment of standard protocols for high and low grading/staging sarcoma patients, mostly with chemotherapy and/or radiotherapy, 50% of treated patients experience relapse episodes. Because of this, in the last 20 years, new therapeutic approaches for sarcoma treatment have been evaluated in preclinical and clinical studies. Among them, antibody-based therapies have been the most studied. Immunoconjugates consist of a carrier portion, frequently represented by an antibody, linked to a toxic moiety, i.e., a drug, toxin, or radionuclide. While the efficacy of immunoconjugates is well demonstrated in the therapy of hematological tumors and more recently also of epithelial ones, their potential as therapeutic agents against sarcomas is still not completely explored. In this paper, we summarize the results obtained with immunoconjugates targeting sarcoma surface antigens, considering both preclinical and clinical studies. To date, the encouraging results obtained in preclinical studies allowed nine immunoconjugates to enter clinical trials, demonstrating the validity of immunotherapy as a promising pharmacological tool also for sarcoma therapy.

Momordica charantia, commonly called bitter melon, is a plant belonging to Cucurbitaceae family known for centuries for its pharmacological activities, and nutritional properties. Due to the presence of many bioactive compounds, some of which possess potent biological actions, this plant is used in folk medicine all over the world for the treatment of different pathologies, mainly diabetes, but also cancer, and other inflammation-associated diseases. It is widely demonstrated that M. charantia extracts contribute in lowering glycaemia in patients affected by type 2 diabetes. However, the majority of existing studies on M. charantia bioactive compounds were performed only on cell lines and in animal models. Therefore, because the real impact of bitter melon on human health has not been thoroughly demonstrated, systematic clinical studies are needed to establish its efficacy and safety in patients. Besides, both in vitro and in vivo studies have demonstrated that bitter melon may also elicit toxic or adverse effects under different conditions. The aim of this review is to provide an overview of anti-inflammatory and anti-neoplastic properties of bitter melon, discussing its pharmacological activity as well as the potential adverse effects. Even if a lot of literature is available about bitter melon as antidiabetic drug, few papers discuss the anti-inflammatory and anti-cancer properties of this plant.

Obesity and related pathologies such as diabetes and metabolic syndrome are associated with chronic inflammation and cancer. The serum level of xanthine oxidoreductase (XOR) is correlated to obesity-associated metabolic disorders. XOR can play a role in the pathogenesis of both metabolic syndrome and cancer through the inflammatory response and the oxidative stress elicited by the products of its activity. The reactive oxygen and nitrogen species and the uric acid derived from XOR concur to the development of hypertension, dyslipidemia and insulin resistance and participate in both cell transformation and proliferation, as well as in the progression and metastasis process. Despite the availability of different drugs to inhibit in vivo XOR activity, the complexity of XOR inhibition effects should be carefully considered before clinical application, save in the case of symptomatic hyperuricemia. Keywords: Cancer risk, Inflammation, Metabolic syndrome, Oncogenesis, Oxidative stress, Xanthine oxidoreductase

The sexually transmitted pathogen Chlamydia trachomatis (CT) is able to replicate and survive in human intestinal epithelial cells, being the gastro-intestinal tract a suitable site of residence for this microorganism. In this context, no detailed information about the mechanisms of cell death in intestinal cell lines after a chlamydial infection is available. The aim of this study was to compare the effect of two different CT serovars (D and L2) on the survival/death of different intestinal cell lines (Caco-2 and COLO-205), using endocervical cells (HeLa) as a reference model of genital infection. Seventy two hours after chlamydial infection at different multiplicity of infection (MOI) levels, the viability of HeLa, Caco-2 and COLO 205 cells was evaluated through dose-response experiments by means of a MTS-based assay. To get deeper insights in the mechanisms of cell death induced by CT, cell viability was assessed in presence of different inhibitors (i.e. pan-caspase inhibitor Z-VAD, necroptosis inhibitor Necrostatin-1, hydrogen peroxide scavenger catalase, caspase-1 inhibitor Ac-YVAD-cmk). Moreover, the activation of effector caspases and the presence of cellular apoptotic/necrotic changes were evaluated at different time points after CT infection. Our results demonstrated that, for both chlamydial serovars, intestinal cell lines are more resistant to CT-induced cell death compared to HeLa, thus representing a suitable 'niche' for chlamydial residence and replication. In literature, apoptosis has been widely described to be the main cell death mechanism elicited by chlamydia infection. However, our data demonstrate that necroptosis plays a relevant role, proceeding in parallel with apoptosis. The protective effect of catalase suggests the involvement of oxidative stress in triggering both cell death pathways. Moreover, we demonstrated that caspase-1 is involved in CT-induced cell death, potentially contributing to host inflammatory response and tissue damage. Cells infected by L2 serovar displayed a higher activation of effector caspases compared to cells infected with serovar D, suggesting a serovar-specific activation of apoptotic pathways and potentially explaining the greater virulence of L serovars. Finally, we found that Chlamydia elicits the early externalization of phosphatidylserine on the external leaflet of plasma membrane independently of caspase activation.

Ribosome-inactivating proteins (RIPs) are plant toxins that irreversibly damage ribosomes and other substrates, thus causing cell death. RIPs are classified in type 1 RIPs, single-chain enzymatic proteins, and type 2 RIPs, consisting of active A chains, similar to type 1 RIPs, linked to lectin B chains, which enable the rapid internalization of the toxin into the cell. For this reason, many type 2 RIPs are very cytotoxic, ricin, volkensin and stenodactylin being the most toxic ones. From the caudex of Adenia kirkii (Mast.) Engl., a new type 2 RIP, named kirkiin, was purified by affinity chromatography on acid-treated Sepharose CL-6B and gel filtration. The lectin, with molecular weight of about 58 kDa, agglutinated erythrocytes and inhibited protein synthesis in a cell-free system at very low concentrations. Moreover, kirkiin was able to depurinate mammalian and yeast ribosomes, but it showed little or no activity on other nucleotide substrates. In neuroblastoma cells, kirkiin inhibited protein synthesis and induced apoptosis at doses in the pM range. The biological characteristics of kirkiin make this protein a potential candidate for several experimental pharmacological applications both alone for local treatments and as component of immunoconjugates for systemic targeting in neurodegenerative studies and cancer therapy.

The senescence process is the result of a series of factors that start from the genetic constitution interacting with epigenetic modifications induced by endogenous and environmental causes and that lead to a progressive deterioration at the cellular and functional levels. One of the main causes of aging is oxidative stress deriving from the imbalance between the production of reactive oxygen (ROS) and nitrogen (RNS) species and their scavenging through antioxidants. Xanthine oxidoreductase (XOR) activities produce uric acid, as well as reactive oxygen and nitrogen species, which all may be relevant to such equilibrium. This review analyzes XOR activity through in vitro experiments, animal studies and clinical reports, which highlight the pro-aging effects of XOR products. However, XOR activity contributes to a regular level of ROS and RNS, which appears essential for the proper functioning of many physiological pathways. This discourages the use of therapies with XOR inhibitors, unless symptomatic hyperuricemia is present.

Stenodactylin is one of the most potent type 2 ribosome-inactivating proteins (RIPs); its high toxicity has been demonstrated in several models both in vitro and in vivo. Due to its peculiarities, stenodactylin could have several medical and biotechnological applications in neuroscience and cancer treatment. In this work, we report the complete amino acid sequence of stenodactylin and 3D structure prediction. The comparison between the primary sequence of stenodactylin and other RIPs allowed us to identify homologies/differences and the amino acids involved in RIP toxic activity. Stenodactylin RNA was isolated from plant caudex, reverse transcribed through PCR and the cDNA was amplificated and cloned into a plasmid vector and further analyzed by sequencing. Nucleotide sequence analysis showed that stenodactylin A and B chains contain 251 and 258 amino acids, respectively. The key amino acids of the active site described for ricin and most other RIPs are also conserved in the stenodactylin A chain. Stenodactylin amino acid sequence shows a high identity degree with volkensin (81.7% for A chain, 90.3% for B chain), whilst when compared with other type 2 RIPs the identity degree ranges from 27.7 to 33.0% for the A chain and from 42.1 to 47.7% for the B chain.

Plants and their corresponding botanical preparations have been used for centuries due to their remarkable potential in both the treatment and prevention of oxidative stress-related disorders. Aging and aging-related diseases, like cardiovascular disease, cancer, diabetes, and neurodegenerative disorders, which have increased exponentially, are intrinsically related with redox imbalance and oxidative stress. Hundreds of biologically active constituents are present in each whole plant matrix, providing promissory bioactive effects for human beings. Indeed, the worldwide population has devoted increased attention and preference for the use of medicinal plants for healthy aging and longevity promotion. In fact, plant-derived bioactives present a broad spectrum of biological effects, and their antioxidant, anti-inflammatory, and, more recently, anti-aging effects, are considered to be a hot topic among the medical and scientific communities. Nonetheless, despite the numerous biological effects, it should not be forgotten that some bioactive molecules are prone to oxidation and can even exert pro-oxidant effects. In this sense, the objective of the present review is to provide a detailed overview of plant-derived bioactives in age-related disorders. Specifically, the role of phytochemicals as antioxidants and pro-oxidant agents is carefully addressed, as is their therapeutic relevance in longevity, aging-related disorders, and healthy-aging promotion. Finally, an eye-opening look into the overall evidence of plant compounds related to longevity is presented.

The castor plant (Ricinus communis L.) has been known since time immemorial in traditional medicine in the pharmacopeia of Mediterranean and eastern ancient cultures. Moreover, it is still used in folk medicine worldwide. Castor bean has been mainly recommended as anti-inflammatory, anthelmintic, anti-bacterial, laxative, abortifacient, for wounds, ulcers, and many other indications. Many cases of human intoxication occurred accidentally or voluntarily with the ingestion of castor seeds or derivatives. Ricinus toxicity depends on several molecules, among them the most important is ricin, a protein belonging to the family of ribosome-inactivating proteins. Ricin is the most studied of this category of proteins and it is also known to the general public, having been used for several biocrimes. This manuscript intends to give the reader an overview of ricin, focusing on the historical path to the current knowledge on this protein. The main steps of ricin research are here reported, with particular regard to its enzymatic activity, structure, and cytotoxicity. Moreover, we discuss ricin toxicity for animals and humans, as well as the relation between bioterrorism and ricin and its impact on environmental toxicity. Ricin has also been used to develop immunotoxins for the elimination of unwanted cells, mainly cancer cells; some of these immunoconjugates gave promising results in clinical trials but also showed critical limitation.

Non-Hodgkin’s lymphomas (NHLs) are a heterogeneous group of different haematological cancers with a wide range of aggressiveness. NHLs represent >80% of lymphomas and the majority of NHLs involve B cells. CD20 represents a good target for NHL immunotherapy because it is largely expressed on B cell NHL and not on B cell precursors and plasma cells. The anti-CD20 monoclonal antibody (mAb) rituximab (RTX) was the first antibody approved by the FDA for lymphoma therapy and has revolutionised B cell lymphoma treatment. Several clinical trials have demonstrated the high efficacy of RTX, resulting in a significant improvement in overall response rates and in NHL patient survival. However, RTX, both as a single agent and in combination with chemotherapy, induces several side-effects and resistance mechanisms. Remarkable efforts have been made to improve RTX efficacy, including conjugation to an active moiety (radionuclide, toxin, enzyme, or drug) and the development of new anti-CD20 mAbs. This review summarises the characteristics of RTX and other anti-CD20 mAbs for NHL treatment; the results of the main clinical trials are reported.

Thirty years ago, the type 1 ribosome-inactivating protein (RIP) saporin-S6 (also known as saporin) was isolated from Saponaria officinalis L. seeds. Since then, the properties and mechanisms of action of saporin-S6 have been well characterized, and it has been widely employed in the construction of conjugates and immunotoxins for different purposes. These immunotoxins have shown many interesting results when used in cancer therapy, particularly in hematological tumors. The high enzymatic activity, stability and resistance to conjugation procedures and blood proteases make saporin-S6 a very useful tool in cancer therapy. High efficacy has been reported in clinical trials with saporin-S6-containing immunotoxins, at dosages that induced only mild and transient side effects, which were mainly fever, myalgias, hepatotoxicity, thrombocytopenia and vascular leak syndrome. Moreover, saporin-S6 triggers multiple cell death pathways, rendering impossible the selection of RIP-resistant mutants. In this review, some aspects of saporin-S6, such as the chemico-physical characteristics, the structural properties, its endocytosis, its intracellular routing and the pathogenetic mechanisms of the cell damage, are reported. In addition, the recent progress and developments of saporin-S6-containing immunotoxins in cancer immunotherapy are summarized, including in vitro and in vivo pre-clinical studies and clinical trials.

Ribosome-inactivating proteins (RIPs) are a family of plant toxins that permanently damage ribosomes and possibly other cellular substrates, thus causing cell death. RIPs are mostly divided in two types: Type 1 RIPs that are single-chain enzymatic proteins, and type 2 RIPs that consist of an active A chain (similar to a type 1 RIP) linked to a B chain with lectin properties. RIP-containing conjugates have been used in many experimental strategies against cancer cells, often showing great efficacy in clinical trials. Saporin-S6, a type 1 RIP extracted from Saponaria officinalis L. seeds, has been extensively utilized to construct anti-cancer conjugates because of its high enzymatic activity, stability and resistance to conjugation procedures, resulting in the efficient killing of target cells. This review summarizes saporin-S6-containing conjugates and their application in cancer therapy, considering in-vitro and in-vivo studies both in animal models and in clinical trials. The review is structured on the basis of the targeting of hematological versus solid tumors and on the antigen recognized on the cell surface.

Bougainvillea (Bougainvillea spectabilis Willd.) is a plant widely used in folk medicine and many extracts from different tissues of this plant have been employed against several pathologies. The observation that leaf extracts of Bougainvillea possess antiviral properties led to the purification and characterization of a protein, named bouganin, which exhibits typical characteristics of type 1 ribosome-inactivating proteins (RIPs). Beyond that, bouganin has some peculiarities, such as a higher activity on DNA with respect to ribosomal RNA, low systemic toxicity, and immunological properties quite different than other RIPs. The sequencing of bouganin and the knowledge of its three-dimensional structure allowed to obtain a not immunogenic mutant of bouganin. These features make bouganin a very attractive tool as a component of immunotoxins (ITs), chimeric proteins obtained by linking a toxin to a carrier molecule. Bouganin-containing ITs showed very promising results in the experimental treatment of both hematological and solid tumors, and one bouganin-containing IT has entered Phase I clinical trial. In this review, we summarize the milestones of the research on bouganin such as bouganin chemico-physical characteristics, the structural properties and de-immunization studies. In addition, the in vitro and in vivo results obtained with bouganin-containing ITs are summarized.

Sporadic Alzheimer’s disease (AD) is the most frequent form of dementia in the elderly. A non-conservative polymorphism in the urokinase-type plasminogen activator gene (PLAU_1=RS2227564) has been analyzed, but data are conflicting on whether it is a risk factor for AD. To clarify whether this genetic variant modifies AD risk in the Italian population, we ran a case–control association study on 192 AD and 126 age-matched controls. We did not find any association between PLAU_1 genotype and AD in the whole AD population, but when we stratified our sample by APOE-ε4 status, we found a significant association between PLAU_1 genotype (C/T+T/T) and APOE-ε4 negative AD subjects (p=0.02, χ2-test). The PLAU_1 genotype did not appear to affect the plasma Aβ42 concentration. Our data support a role for PLAU_1 as an independent genetic risk factor for AD in the Italian population for those subjects who do not have the APOE-ε4 allele.

Depression is common in the elderly. The role of genetic and environmental factors in modulating depressive symptoms is not clear.We evaluated the influence of serotonin transporter gene polymorphisms and recent adverse life events on depressive symptoms in an elderly Italian population. We used data from "InveCe.Ab", a population-based study of 1321 subjects aged 70-74 years. We used the 15-item Geriatric Depression Scale (GDS) to assess depressive symptoms-a GDS score ≥5 points (GDS≥5) indicated the presence of clinically relevant symptoms-and performed 5-HTTLPR and rs25531 genotyping to obtain the triallelic polymorphism of the serotonin transporter. We used the Geriatric Adverse Life Events Scale to measure adverse life events, and logistic regression models to evaluate the role of genotype and recent adverse life events in depressive symptoms, controlling for potential confounders and independent predictors.Two hundred subjects (15.76%) had a GDS≥5. The 5-HTTLPR triallelic polymorphism was significantly associated with GDS≥5. Only S'S' carriers showed an increased risk of depressive symptoms (ORadj = 1.81, p = .022); one extra adverse life event increased this risk by 14% (p = .061) independently of genotype. Other factors significantly related to GDS≥5 were: female gender (ORadj = 2.49, p < .001), age (ORadj = 1.19, p = .007), a history of depression (ORadj = 4.73, p < .001), and comorbidity (ORadj = 1.23, p = .001). One extra adverse life event increased the risk of depressive symptoms by 57% (p = .005) only in the L'L' carriers, while antidepressant intake was directly related to GDS≥5 in the L'S' carriers (ORadj = 2.46, p = .036) and borderline significant in the S'S' carriers (ORadj = 2.41, p = .081).The S'S' genotype and recent exposure to adverse life events were independently associated with depressive symptoms. The S'S' genotype, compared with the environment, exerted a predominant effect on depressive symptoms, suggesting that it reduces the efficacy of antidepressant therapy. We conclude that genetics may be an important risk factor for depressive symptoms in late adulthood.

Immunotoxins (ITs) are hybrid proteins combining the binding specificity of antibodies with the cytocidal properties of toxins. They represent a promising approach to lymphoma therapy. The cytotoxicity of two immunotoxins obtained by chemical conjugation of the plant toxin saporin-S6 with the anti-CD20 chimeric antibody rituximab and the anti-CD22 murine antibody OM124 were evaluated on the CD20-/CD22-positive cell line Raji. Both ITs showed strong cytotoxicity for Raji cells, but the anti-CD22 IT was two logs more efficient in killing, probably because of its faster internalization. The anti-CD22 IT gave slower but greater caspase activation than the anti-CD20 IT. The cytotoxic effect of both immunotoxins can be partially prevented by either the pan-caspase inhibitor Z-VAD or the necroptosis inhibitor necrostatin-1. Oxidative stress seems to be involved in the cell killing activity of anti-CD20 IT, as demonstrated by the protective role of the H2O2 scavenger catalase, but not in that of anti-CD22 IT. Moreover, the IT toxicity can be augmented by the contemporary administration of other chemotherapeutic drugs, such as PS-341, MG-132, and fludarabine. These results contribute to the understanding of the immunotoxin mechanism of action that is required for their clinical use, either alone or in combination with other drugs.

The enzymes called ribosome‐inactivating proteins (RIPs) that are able to depurinate nucleic acids and arrest vital cellular functions, including protein synthesis, are still a frontline research field, mostly because of their promising medical applications. The contributions of Stirpe to the development of these studies has been one of the most relevant. After a short biographical introduction, an overview is offered of the main results obtained by his investigations during last 55 years on his main research lines: hyperuricaemia, xanthine oxidoreductase and RIPs.

Palmitic acid is known to be apoptotic for nervous cells but no data are available on membrane lipidome transformations occurring during its supplementation, although membrane lipids are clearly involved in the apoptotic signaling cascade. NB100 neuroblastoma cells were supplemented with palmitic acid and membrane fatty acids were isolated, derivatized and analysed by gas chromatography at defined time intervals. Parallely, cell viability, morphology, apoptosis, cPLA(2) and caspase activations were checked. Interestingly, under 150 µM supplementation the incorporation of palmitic acid was accompanied by the specific release of arachidonic acid. This event was timely correlated with cPLA(2) and caspases activations, and the time window of 60 minutes was envisaged for crucial membrane lipidome changes. The simultaneous addition of 50 µM oleic, 50 µM arachidonic and 150 µM palmitic acids to the cell cultures influenced membrane changes with suppression of caspase activation and maintenance of cell viability. These results highlight the role of the membrane asset with fatty acid remodeling and suggest the potential of lipid-based strategies for influencing cell response and fate in human diseases, such as neurodegenerative disorders or tumours.

This review provides a historical overview of the research on plant ribosome-inactivating proteins (RIPs), starting from the first studies at the end of eighteenth century involving the purification of abrin and ricin, as well as the immunological experiments of Paul Erlich. Interest in these plant toxins was revived in 1970 by the observation of their anticancer activity, which has given rise to a large amount of research contributing to the development of various scientific fields. Biochemistry analyses succeeded in identifying the enzymatic activity of RIPs and allowed for a better understanding of the ribosomal machinery. Studies on RIP/cell interactions were able to detail the endocytosis and intracellular routing of ricin, thus increasing our knowledge of how cells handle exogenous proteins. The identification of new RIPs and the finding that most RIPs are single-chain polypeptides, together with their genetic sequencing, has aided in the development of new phylogenetic theories. Overall, the biological properties of these proteins, including their abortifacient, anticancer, antiviral and neurotoxic activities, suggest that RIPs could be utilized in agriculture and in many biomedical fields, including clinical drug development.

Ribosome-inactivating proteins (RIPs) are enzymes that deadenylate nucleic acids and are broadly distributed in the plant kingdom. Many plants that contain RIPs are listed in the pharmacopoeias of folk medicine all over the world, mostly because of their toxicity. This review analyses the position occupied in traditional medicine by plants from which RIPs have been isolated. The overview starts from the antique age of the Mediterranean area with ancient Egypt, followed by the Greek and Roman classic period. Then, the ancient oriental civilizations of China and India are evaluated. More recently, Unani medicine and European folk medicine are examined. Finally, the African and American folk medicines are taken into consideration. In conclusion, a list of RIP-expressing plants, which have been used in folk medicine, is provided with the geographical distribution and the prescriptions that are recommended by traditional healers. Some final considerations are provided on the present utilization of such herbal treatments, both in developing and developed countries, often in the absence of scientific validation. The most promising prospect for the medicinal use of RIP-expressing plants is the conjugation of purified RIPs to antibodies that recognise tumour antigens for cancer therapy.

Immunotoxins are chimeric proteins obtained by linking a toxin to either an intact antibody or an antibody fragment. Conjugation can be obtained by chemical or genetic engineering, where the latter yields recombinant conjugates. An essential requirement is that the target molecule recognized by the antibody is confined to the cell population to be deleted, or at least that it is not present on stem cells or other cell types essential for the organism’s survival. Hundreds of different studies have demonstrated the potential for applying immunotoxins to many models in pre-clinical studies and in clinical trials. Immunotoxins can be theoretically used to eliminate any unwanted cell responsible for a pathological condition. The best results have been obtained in cancer therapy, especially in hematological malignancies. Among plant toxins, the most frequently employed to generate immunotoxins are ribosome-inactivating proteins, the most common being ricin. This review summarizes the various approaches and results obtained in the last four decades by researchers in the field of plant toxin-based immunotoxins for cancer therapy.

The anti-CD20 mAb Rituximab has revolutionized lymphoma therapy, in spite of a number of unresponsive or relapsing patients. Immunotoxins, consisting of toxins coupled to antibodies, are being investigated for their potential ability to augment Rituximab efficacy. Here, we compare the anti-tumor effect of high- and low-molecular-weight Rituximab/saporin-S6 immunotoxins, named HMW-IT and LMW-IT, respectively. Saporin-S6 is a potent and stable plant enzyme belonging to ribosome-inactivating proteins that causes protein synthesis arrest and consequent cell death. Saporin-S6 was conjugated to Rituximab through an artificial disulfide bond. The inhibitory activity of HMW-IT and LMW-IT was evaluated on cell-free protein synthesis and in two CD20+ lymphoma cell lines, Raji and D430B. Two different conjugates were separated on the basis of their molecular weight and further characterized. Both HMW-IT (dimeric) and LMW-IT (monomeric) maintained a high level of enzymatic activity in a cell-free system. HMW-IT, thanks to a higher toxin payload and more efficient antigen capping, showed stronger in vitro anti-tumor efficacy than LMW-IT against lymphoma cells. Dimeric HMW-IT can be used for lymphoma therapy at least for ex vivo treatments. The possibility of using HMW-IT augments the yield in immunotoxin preparation and allows the targeting of antigens with low internalization rates.

Alzheimer’s disease (AD) is a neurodegenerative disorder whose clinical onset is mainly characterized by memory loss. During AD progression, behavioral and psychological symptoms of dementia (BPSD) frequently occur. In this paper we evaluated the association between AD and the short/long (S/L) functional polymorphism of the promoter region of the 5-hydroxytryptamine (5-HT) transporter gene (SLC6A4). The S-allele shows a 2-fold reduced transcriptional rate, causing an imbalance in 5-HT intracellular availability that might in turn trigger behavioral and cognitive alterations. We also genotyped the SLC6A4 promoter functional variant rs25531 (A→G). By comparing the genotypic and allelic frequencies in an Italian population of 235 AD and 207 controls, we found an association between 5-HTTLPR and AD (odds ratio for the L-allele versus the S-allele: 0.74, associated P value = .03), while no difference was found for the rs25531. A meta-analysis of studies in Italy assessing 5-HTTLPR and AD risk gave an estimation of odds ratio for the L-allele versus the S-allele of 0.85 (associated P value = .08). Overall, our findings are not supportive of a large genetic effect of the explored polymorphisms on AD risk.

BACKGROUND:Parkinson's disease (PD) is a neurodegenerative pathology whose molecular etiopathogenesis is not known. Novel contributions have come from familial forms of PD caused by alterations in genes with apparently unrelated physiological functions. The gene coding for alpha-synuclein (alpha-syn) (PARK1) has been investigated as alpha-syn is located in Lewy bodies (LB), intraneuronal inclusions in the substantia nigra (SN) of PD patients. A-syn has neuroprotective chaperone-like and antioxidant functions and is involved in dopamine storage and release. DJ-1 (PARK7), another family-PD-linked gene causing an autosomal recessive form of the pathology, shows antioxidant and chaperone-like activities too. METHODOLOGY/PRINCIPAL FINDINGS:The present study addressed the question whether alpha-syn and DJ-1 interact functionally, with a view to finding some mechanism linking DJ-1 inactivation and alpha-syn aggregation and toxicity. We developed an in vitro model of alpha-syn toxicity in the human neuroblastoma cell line SK-N-BE, influencing DJ-1 and alpha-syn intracellular concentrations by exogenous addition of the fusion proteins TAT-alpha-syn and TAT-DJ-1; DJ-1 was inactivated by the siRNA method. On a micromolar scale TAT-alpha-syn aggregated and triggered neurotoxicity, while on the nanomolar scale it was neuroprotective against oxidative stress (induced by H(2)O(2) or 6-hydroxydopamine). TAT-DJ-1 increased the expression of HSP70, while DJ-1 silencing made SK-N-BE cells more susceptible to oxidative challenge, rendering TAT-alpha-syn neurotoxic at nanomolar scale, with the appearance of TAT-alpha-syn aggregates. CONCLUSION/SIGNIFICANCE:DJ-1 inactivation may thus promote alpha-syn aggregation and the related toxicity, and in this model HSP70 is involved in the antioxidant response and in the regulation of alpha-syn fibril formation.