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Abstract: Intravenous plasminogen replacement therapy for patients with plasminogen deficiency type 1 (hypoplasminogenemia) was recently approved for marketing in the US. In this case report, the authors describe a 33-year-old man with hypoplasminogenemia who developed nonhealing postsurgical wounds following trauma to his right hand despite receiving standard treatment for 4 months. The patient was enrolled in a compassionate-use protocol with intravenous plasminogen replacement therapy and experienced prompt resolution of surgical wounds. He was the first human patient to receive replacement therapy with plasminogen, human-tvmh in the US and first to demonstrate cutaneous wound healing in addition to resolution of ligneous lesions attributable to plasminogen deficiency type 1., (Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.)

Aim: An open-label phase 2/3 study of plasminogen, human-tvmh administered intravenously in paediatric and adult subjects with type 1 plasminogen deficiency was conducted. Interim data was previously reported. The final data on 15 subjects who completed the study up to a maximum of 124 weeks are reported here., Methods: The primary objectives were to evaluate efficacy of plasminogen replacement therapy on clinically evident or visible lesions during 48 weeks of dosing and to achieve an increase in trough plasminogen activity levels by at least an absolute 10% above baseline during 12 weeks of treatment., Results: The primary efficacy endpoint was achieved, as 100% of subjects (n = 11) with visible and assessable non-visible lesions at baseline demonstrated ≥ 50% improvement after 48 weeks of study drug treatment with plasminogen, human-tvmh. All subjects achieved the targeted ≥ 10% increase in trough plasminogen activity above baseline through Week 12. Plasminogen, human-tvmh at a dose of 6.6 mg/kg administered every 2-5 days for 48 weeks and every 1-7 days for up to 124 weeks was well tolerated., Conclusion: This study provides additional evidence regarding the long-term safety and clinical utility of replacement therapy with human plasminogen for the treatment of children and adults with type 1 plasminogen deficiency. Plasminogen, human-tvmh received marketing approval on June 4, 2021. This trial was registered at www., Clinicaltrials: gov as #NCT02690714., (© 2023 The Authors. Haemophilia published by John Wiley & Sons Ltd.)

Acute respiratory distress syndrome (ARDS) is a severe complication of lung injuries, commonly associated with bacterial, fungal and viral infections, including SARS-CoV-2 viral infections. ARDS is strongly correlated with patient mortality and its clinical management is very complex, with no effective treatment presently available. ARDS involves severe respiratory failure, fibrin deposition in both airways and lung parenchyma, with the development of an obstructing hyaline membrane drastically limiting gas exchange. Moreover, hypercoagulation is related to deep lung inflammation, and a pharmacological action toward both aspects is expected to be beneficial. Plasminogen (PLG) is a main component of the fibrinolytic system playing key roles in various inflammation regulatory processes. The inhalation of PLG has been proposed in the form of the off-label administration of an eyedrop solution, namely, a plasminogen-based orphan medicinal product (PLG-OMP), by means of jet nebulisation. Being a protein, PLG is susceptible to partial inactivation under jet nebulisation. The aim of the present work is to demonstrate the efficacy of the mesh nebulisation of PLG-OMP in an in vitro simulation of clinical off-label administration, considering both the enzymatic and immunomodulating activities of PLG. Biopharmaceutical aspects are also investigated to corroborate the feasibility of PLG-OMP administration by inhalation. The nebulisation of the solution was performed using an Aerogen ® Solo TM vibrating-mesh nebuliser. Aerosolised PLG showed an optimal in vitro deposition profile, with 90% of the active ingredient impacting the lower portions of a glass impinger. The nebulised PLG remained in its monomeric form, with no alteration of glycoform composition and 94% of enzymatic activity maintenance. Activity loss was observed only when PLG-OMP nebulisation was performed under simulated clinical oxygen administration. In vitro investigations evidenced good penetration of aerosolised PLG through artificial airway mucus, as well as poor permeation across an Air-Liquid Interface model of pulmonary epithelium. The results suggest a good safety profile of inhalable PLG, excluding high systemic absorption but with good mucus diffusion. Most importantly, the aerosolised PLG was capable of reversing the effects of an LPS-activated macrophage RAW 264.7 cell line, demonstrating the immunomodulating activity of PLG in an already induced inflammatory state. All physical, biochemical and biopharmaceutical assessments of mesh aerosolised PLG-OMP provided evidence for its potential off-label administration as a treatment for ARDS patients.

Background: Pronectins™ are a new class of fibronectin-3-domain 14th-derived (14Fn3) antibody mimics that can be engineered as bispecific T cell engager (BTCE) to redirect immune effector cells against cancer. We describe here the in vitro and in vivo activity of a Pronectin™ AXL-targeted first-in-class bispecific T cell engager (pAXLxCD3ε) against Epithelial Ovarian Cancer (EOC)., Methods: pAXLxCD3ε T-cell mediated cytotoxicity was evaluated by flow cytometry and bioluminescence. pAXLxCD3ε mediated T-cell infiltration, activation and proliferation were assessed by immunofluorescence microscopy and by flow cytometry. Activity of pAXLxCD3ε was also investigated in combination with poly-ADP ribose polymerase inhibitors (PARPi). In vivo antitumor activity of pAXLxCD3ε was evaluated in immunocompromised (NSG) mice bearing intraperitoneal or subcutaneous EOC xenografts and immunologically reconstituted with human peripheral blood mononuclear cells (PBMC)., Results: pAXLxCD3ε induced dose-dependent cytotoxicity by activation of T lymphocytes against EOC cells, regardless of their histologic origin. The addition of PARPi to cell cultures enhanced pAXLxCD3ε cytotoxicity. Importantly, in vivo, pAXLxCD3ε was highly effective against EOC xenografts in two different NSG mouse models, by inhibiting the growth of tumor cells in ascites and subcutaneous xenografts. This effect translated into a significantly prolonged survival of treated animals., Conclusion: pAXLxCD3ε is an active therapeutics against EOC cells providing a rational for its development as a novel agent in this still incurable disease. The preclinical validation of a first-in-class agent opens the way to the development of a new 14Fn3-based scaffold platform for the generation of innovative immune therapeutics against cancer., (© 2023. The Author(s).)

Sarcomas are heterogeneous malignancies with limited therapeutic options and a poor prognosis. We developed an innovative immunotherapeutic agent, a first-in-class Pronectin™-based Bispecific T-Cell Engager (pAXL×CD3ε), for the targeting of AXL, a TAM family tyrosine kinase receptor highly expressed in sarcomas. AXL expression was first analyzed by flow cytometry, qRT-PCR, and Western blot on a panel of sarcoma cell lines. The T-cell-mediated pAXL×CD3ε cytotoxicity against sarcoma cells was investigated by flow cytometry, luminescence assay, and fluorescent microscopy imaging. The activation and degranulation of T cells induced by pAXL×CD3ε were evaluated by flow cytometry. The antitumor activity induced by pAXL×CD3ε in combination with trabectedin was also investigated. In vivo activity studies of pAXL×CD3ε were performed in immunocompromised mice (NSG), engrafted with human sarcoma cells and reconstituted with human peripheral blood mononuclear cells from healthy donors. Most sarcoma cells showed high expression of AXL. pAXL×CD3ε triggered T-lymphocyte activation and induced dose-dependent T-cell-mediated cytotoxicity. The combination of pAXL×CD3ε with trabectedin increased cytotoxicity. pAXL×CD3ε inhibited the in vivo growth of human sarcoma xenografts, increasing the survival of treated mice. Our data demonstrate the antitumor efficacy of pAXL×CD3ε against sarcoma cells, providing a translational framework for the clinical development of pAXL×CD3ε in the treatment of human sarcomas, aggressive and still-incurable malignancies.

A highly specific AXL-receptor targeted family of non-immunoglobulin, single domain protein binders (Pronectins™) have been isolated from three (3) synthetic libraries that employ the human scaffold of the 14th domain of Fibronectin III (14FN3) and evolutionary CDRs diversity of over 25 billion loop sequences. The three libraries, each containing diversity in two loops, were designed to expand upon a human database of more than 6000 natural scaffold sequences and approximately 3000 human loop sequences. We used a bioinformatic-based approach to maximize "human" amino acid loop diversity and minimize or prevent altogether CDR immunogenicity created by the use of mutagenesis processes to generate diversity. A combination of phage display and yeast display was used to isolate 59 AXL receptor targeted Pronectins with KD ranging between 2 and 100 nM. FACS analysis with tumor cells over-expressing AXL and the use of an AXL knock-out cell line allowed us to identify Pronectin candidates with exquisite specificity for AXL receptor. Based upon several in vitro cell-based tests, we selected the best candidate, AXL54, to further characterize its in vitro cancer cells killing activity. Finally, AXL54 was used to produce the first bi-specific T cell engager protein (AXL54 [Pronectin]-linker-scFV CD3), a "new in class" protein for further testing of its anti-tumor activity in vitro and in vivo.

Most chronic illnesses are caused by the biological reaction to an injury, rather than the initial injury or the injurious agent itselves as in neurodegeneration. With respect to this, notable attention is emerging on the therapeutic effects of dietary polyphenols for human health, able to counteract and neutralize oxidative stress and inflammatory processes involved in the etiopathogenesis of major neurodegenerative disorders, including Alzheimer's disease and Parkinson's disease. The acquired concept that cellular stress at low doses induces neuroprotective responses against degenerative processes is a frontier area of the neurobiological research focusing on the development of novel preventive and therapeutic interventions for neurodegenerative disorders. Notably, basal levels of prooxidant species are essential to promote adaptive redox cellular responses including vitagenes, tightly correlated to cell survival against age-related diseases. In this paper we discuss the concept of cellular stress response and hormesis and its applications to the field of neuroprotection and the potential therapeutic support provided by olive polyphenols, in particular hydroxytyrosol (HT)-rich aqueous olive pulp extract (Hidrox), as a pivotal activator of Nrf2 pathway and related vitagenes, and inhibitor of Keap1-Nrf2 interaction.Olive polyphenols are considered potential pharmacological modulators of neuroinflammation by upregulation of the Keap1/Nfr2/ARE pathway thus providing a strong rationale for treating neurodegenerative disorders., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Fibromyalgia (FM) is a chronic condition characterized by persistent widespread pain that negatively affects the quality of life of patients. The WNT/β-catenin signaling pathway seems to be involved in central sensitization and different pain states. The objective of this study was to investigate the beneficial effects of a new compound called Hidrox ® (HD), containing 40-50% hydroxytyrosol, in counteracting the pain associated with FM. An FM-like model was induced in rats by subcutaneous injections of reserpine (1 mg/kg) for three consecutive days. Later, HD (10 mg/kg) was administered orally to the animals for seven days. Reserpine injections induced WNT/β-catenin pathway activation, release of pro-inflammatory mediators as well as a significant increase in oxidative stress. Daily treatment with HD was able to modulate the WNT/β-catenin and Nrf2 pathways and consequently attenuate the behavioral deficits and microglia activation induced by reserpine injection. These results indicate that nutritional consumption of HD can be considered as a new therapeutic approach for human FM.

Polyphenols are chemopreventive through the induction of nuclear factor erythroid 2 related factor 2 (Nrf2)-mediated proteins and anti-inflammatory pathways. These pathways, encoding cytoprotective vitagenes, include heat shock proteins, such as heat shock protein 70 (Hsp70) and heme oxygenase-1 (HO-1), as well as glutathione redox system to protect against cancer initiation and progression. Phytochemicals exhibit biphasic dose responses on cancer cells, activating at low dose, signaling pathways resulting in upregulation of vitagenes, as in the case of the Nrf2 pathway upregulated by hydroxytyrosol (HT) or curcumin and NAD/NADH-sirtuin-1 activated by resveratrol. Here, the importance of vitagenes in redox stress response and autophagy mechanisms, as well as the potential use of dietary antioxidants in the prevention and treatment of multiple types of cancer are discussed. We also discuss the possible relationship between SARS-CoV-2, inflammation and cancer, exploiting innovative therapeutic approaches with HT-rich aqueous olive pulp extract (Hidrox®), a natural polyphenolic formulation, as well as the rationale of Vitamin D supplementation. Finally, we describe innovative approaches with organoids technology to study human carcinogenesis in preclinical models from basic cancer research to clinical practice, suggesting patient-derived organoids as an innovative tool to test drug toxicity and drive personalized therapy., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Interstitial cystitis/painful bladder syndrome (IC/PBS) is a chronic bladder condition characterized by frequent urination, inflammation, oxidative stress, and pain. The aim of the study was to evaluate the anti-inflammatory and antioxidant effects of an oral administration of Hidrox ® (10 mg/kg) in the bladder and spinal cord in a rodent model of IC/BPS. The chronic animal model of cystitis was induced by repeated intraperitoneal injections of cyclophosphamide (CYP) for five consecutive days. Treatment with Hidrox ® began on the third day of the CYP injection and continued until the 10th day. CYP administration caused macroscopic and histological bladder changes, inflammatory infiltrates, increased mast cell numbers, oxidative stress, decreased expression of the tight endothelial junction (e.g., zonula occludens-1 (ZO-1) and occludin), and bladder pain. Treatment with Hidrox ® was able to improve CYP-induced inflammation and oxidative stress via the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) pathway. It was also able to reduce bladder pain which was aggravated by the activation of neuroinflammation in the central nervous system. In particular, Hidrox ® reduced the brain-derived neurotrophic factor (BDNF), as well as the activation of astrocytes and microglia, consequently reducing mechanical allodynia. These results indicate that nutritional consumption of Hidrox ® can be considered as a new therapeutic approach for human cystitis, increasing the conceivable potential of a significant improvement in the quality of life associated with a lowering of symptom intensity in patients with IC/BPS.

Traumatic brain injuries (TBI) are a serious public-health problem. Furthermore, subsequent TBI events can compromise TBI patients' quality of life. TBI is linked to a number of long- and short-term complications such as cerebral atrophy and risk of developing dementia and Alzheimer's Disease (AD). Following direct TBI damage, oxidative stress and the inflammatory response lead to tissue injury-associated neurodegenerative processes that are characteristic of TBI-induced secondary damage. Hidrox ® showed positive effects in preclinical models of toxic oxidative stress and neuroinflammation; thus, the aim of this study was to evaluate the effect of Hidrox ® administration on TBI-induced secondary injury and on the propagation of the AD-like neuropathology. Hidrox ® treatment reduced histological damage after controlled cortical impact. Form a molecular point of view, hydroxytyrosol is able to preserve the cellular redox balance and protein homeostasis by activating the Nrf2 pathway and increasing the expression of phase II detoxifying enzymes such as HO-1, SOD, Catalase, and GSH, thus counteracting the neurodegenerative damage. Additionally, Hidrox ® showed anti-inflammatory effects by reducing the activation of the NFkB pathway and related cytokines overexpression. From a behavioral point of view, Hidrox ® treatment ameliorated the cognitive dysfunction and memory impairment induced by TBI. Additionally, Hidrox ® was associated with a significant increased number of hippocampal neurons in the CA3 region, which were reduced post-TBI. In particular, Hidrox ® decreased AD-like phenotypic markers such as ß-amyloid accumulation and APP and p-Tau overexpression. These findings indicate that Hidrox ® could be a valuable treatment for TBI-induced secondary injury and AD-like pathological features.

Background: Every year, men use cyclophosphamide to treat various cancers and autoimmune diseases. On the one hand, this chemotherapy often has the beneficial effect of regressing the tumor, but on the other hand, it leads to infertility due to excessive oxidative stress and apoptosis in the testes caused by its metabolite, acrolein., Methods: The objective of this study was to evaluate the beneficial power of a new compound called Hidrox ® , containing 40-50% hydroxytyrosol, in counteracting the damage related to fertility induced by cyclophosphamide. The study was conducted using a single intraperitoneal injection of cyclophosphamide at a dose of 200 mg/kg b.w, in distilled water at 10 mL/kg b.w. The treatment was administered via the oral administration of Hidrox ® at a dose of 50 mg/kg., Results: Our study confirms that the use of cyclophosphamide causes a series of sperm and histological alterations strongly connected with oxidative stress, lipid peroxidation, and apoptosis., Conclusion: Our results demonstrate for the first time that Hidrox ® protects testes from CYP-induced alterations by the modulation of physiological antioxidant defenses.

Endometriosis is a gynecological and painful condition affecting women of reproductive age. It is characterized by dysfunctional endometrium-like implants outside of the uterine cavity. The purpose of this study was to evaluate the effects of Hidrox ® , an aqueous extract of olive pulp containing hydroxytyrosol, on endometriotic lesions associated with pro-oxidative alterations and pain-like behaviors. Endometriosis was induced by intraperitoneal injection of uterine fragments, and Hidrox ® was administered daily. At the end of the 14-day treatment, behavioral alterations were assessed and hippocampal tissues were collected. Laparotomy was performed, and the endometrial implants were harvested for histological and biochemical analysis. Hidrox ® treatment reduced endometriotic implant area, diameter and volumes. Vehicle-treated rats showed lesional fibrosis, epithelial-mesenchymal transition and fibroblast-myofibroblast transdifferentiation, angiogenesis and pro-oxidative alterations in the peritoneal cavity. Hidrox ® treatment reduced the aniline blue-stained area, α-smooth muscle actin (α-sma) and CD34 positive expressions. Moreover, it reduced mast cell recruitment into the lesions, myeloperoxidase activity and lipid peroxidation and increased superoxide dismutase (SOD) activity and glutathione levels in the endometrial explants. In the peritoneal fluid, Hidrox ® treatment reduced interleukin (IL)-1β, IL2, IL6, tumor necrosis factor-α (TNF-α) and vascular endothelial grow factor (VEGF) levels increased by the disease. Hidrox ® administration also reduced peripheral and visceral sensibility as shown by the behavioral tests (open field test, hot plate test, elevated plus maze test and acetic-acid-induced abdominal contractions). Animals treated with Hidrox ® also showed reduced blood-brain barrier permeability and mast cell infiltration in the hippocampus, as well as astrocyte and microglia activation and brain oxidative status restoring brain-derived neurotrophic factor (BDNF) protein expression and increasing Nuclear factor erythroid 2-related factor 2 (Nfr2) nuclear translocation. In conclusion, Hidrox ® displayed potential ameliorative effects on endometriotic implants and related pain-induced behaviors due to its potent antioxidative properties.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally. Although measures to control SARS-CoV-2, namely, vaccination, medication, and chemical disinfectants are being investigated, there is an increase in the demand for auxiliary antiviral approaches using natural compounds. Here we have focused on hydroxytyrosol (HT)-rich aqueous olive pulp extract (HIDROX ® ) and evaluated its SARS-CoV-2-inactivating activity in vitro. We showed that the HIDROX solution exhibits time- and concentration-dependent SARS-CoV-2-inactivating activities, and that HIDROX has more potent virucidal activity than pure HT. The evaluation of the mechanism of action suggested that both HIDROX and HT induced structural changes in SARS-CoV-2, which changed the molecular weight of the spike proteins. Even though the spike protein is highly glycosylated, this change was induced regardless of the glycosylation status. In addition, HIDROX or HT treatment disrupted the viral genome. Moreover, the HIDROX-containing cream applied on film showed time- and concentration-dependent SARS-CoV-2-inactivating activities. Thus, the HIDROX-containing cream can be applied topically as an antiviral hand cream. Our findings suggest that HIDROX contributes to improving SARS-CoV-2 control measures.

The SARS-CoV-2 infection is associated with pulmonary coagulopathy, which determines the deposition of fibrin in the air spaces and lung parenchyma. The resulting lung lesions compromise patient pulmonary function and increase mortality, or end in permanent lung damage for those who have recovered from the COVID-19 disease. Therefore, local pulmonary fibrinolysis can be efficacious in degrading pre-existing fibrin clots and reducing the conversion of lung lesions into lasting scars. Plasminogen is considered a key player in fibrinolysis processes, and in view of a bench-to-bedside translation, we focused on the aerosolization of an orphan medicinal product (OMP) for ligneous conjunctivitis: human plasminogen (PLG-OMP) eye drops. As such, the sterile and preservative-free solution guarantees the pharmaceutical quality of GMP production and meets the Ph. Eur. requirements of liquid preparations for nebulization. PLG-OMP aerosolization was evaluated both from technological and stability viewpoints, after being submitted to either jet or ultrasonic nebulization. Jet nebulization resulted in a more efficient delivery of an aerosol suitable for pulmonary deposition. The biochemical investigation highlighted substantial protein integrity maintenance with the percentage of native plasminogen band > 90%, in accordance with the quality specifications of PLG-OMP. In a coherent way, the specific activity of plasminogen is maintained within the range 4.8-5.6 IU/mg (PLG-OMP pre-nebulization: 5.0 IU/mg). This is the first study that focuses on the technological and biochemical aspects of aerosolized plasminogen, which could affect both treatment efficacy and clinical dosage delivery. Increasing evidence for the need of local fibrinolytic therapy could merge with the availability of PLG-OMP as an easy handling solution, readily aerosolizable for a fast translation into an extended clinical efficacy assessment in COVID-19 patients.

Background: In developed countries, the extension of human life is increasingly accompanied by a progressive increase in neurodegenerative diseases, most of which do not yet have effective therapy but only symptomatic treatments. In recent years, plant polyphenols have aroused considerable interest in the scientific community. The mechanisms currently hypothesized for the pathogenesis of Parkinson's disease (PD) are neuroinflammation, oxidative stress and apoptosis. Hydroxytyrosol (HT), the main component of Hidrox ® (HD), has been shown to have some of the highest free radical evacuation and anti-inflammatory activities. Here we wanted to study the role of HD on the neurobiological and behavioral alterations induced by rotenone., Methods: A study was conducted in which mice received HD (10 mg/kg, i.p.) concomitantly with rotenone (5 mg/kg, o.s.) for 28 days., Results: Locomotor activity, catalepsy, histological damage and several characteristic markers of the PD, such as the dopamine transporter (DAT) content, tyrosine hydroxylase (TH) and accumulation of α-synuclein, have been evaluated. Moreover, we observed the effects of HD on oxidative stress, neuroinflammation, apoptosis and inflammasomes. Taken together, the results obtained highlight HD's ability to reduce the loss of dopaminergic neurons and the damage associated with it by counteracting the three main mechanisms of PD pathogenesis., Conclusion: HD is subject to fewer regulations than traditional drugs to improve patients' brain health and could represent a promising nutraceutical choice to prevent PD.

Parkinson's disease (PD) is the second most prevalent late-age onset neurodegenerative disorder, affecting 1% of the population after the age of about 60 years old and 4% of those over 80 years old, causing motor impairments and cognitive dysfunction. Increasing evidence indicates that Mediterranean diet (MD) exerts beneficial effects in maintaining health, especially during ageing and by the prevention of neurodegenerative disorders. In this regard, olive oil and its biophenolic constituents like hydroxytyrosol (HT) have received growing attention in the past years. Thus, in the current study we test the health-promoting effects of two hydroxytyrosol preparations, pure HT and Hidrox ® (HD), which is hydroxytyrosol in its "natural" environment, in the established invertebrate model organism Caenorhabditis elegans . HD exposure led to much stronger beneficial locomotion effects in wild type worms compared to HT in the same concentration. Consistent to this finding, in OW13 worms, a PD-model characterized by α-synuclein expression in muscles, HD exhibited a significant higher effect on α-synuclein accumulation and swim performance than HT, an effect partly confirmed also in swim assays with the UA44 strain, which features α-synuclein expression in DA-neurons. Interestingly, beneficial effects of HD and HT treatment with similar strength were detected in the lifespan and autofluorescence of wild-type nematodes, in the neuronal health of UA44 worms as well as in the locomotion of rotenone-induced PD-model. Thus, the hypothesis that HD features higher healthspan-promoting abilities than HT was at least partly confirmed. Our study demonstrates that HD polyphenolic extract treatment has the potential to partly prevent or even treat ageing-related neurodegenerative diseases and ageing itself. Future investigations including mammalian models and human clinical trials are needed to uncover the full potential of these olive compounds.

Anti-inhibitor coagulant complex (AICC), an activated prothrombin complex concentrate, has been available for the treatment of patients with inhibitors since 1977, and thromboembolic events (TEEs) have been reported after infusion of AICC in patients with congenital or acquired hemophilia. With the aim of estimating the TEE incidence rate (IR) related to AICC exposure in these patients, a systematic review of the literature was carried out in Medline, according to PRISMA guidelines, from inception date to March 2017. The IR of TEEs was estimated through a meta-analytic approach by using a generalized linear mixed model based on a Poisson distribution. Thirty-nine studies were included (1980-2016). Overall, 46 TEEs were reported; of these, 13 were reported as disseminated intravascular coagulations, 11 as myocardial infarctions, and 3 as thrombotic cerebrovascular accidents. The pooled TEE IR was 2.87 (95% confidence interval [CI], 0.32-25.40) per 100 000 AICC infusions (5.42 in retrospective studies [95% CI, 0.92-31.82]; 1.09 in prospective studies [95% CI, 0.01-238.77]). The TEE rate was 5.09 (95% CI, 0.01-1795.60) per 100 000 AICC infusions administered on demand, whereas no TEEs were reported with prophylaxis. Interestingly, the estimated IR in patients with congenital hemophilia was <0.01 per 100 000 infusions. These findings provide robust evidence of safety of AICC over almost 40 years of published studies., Competing Interests: Conflict-of-interest disclosure: P.A.C. received a research grant from Baxalta, now part of Shire, and speaking honoraria from Pfizer. L.G.M. reports personal fees from Pfizer and Bayer Healthcare. A.G. and R.C. are employed by Shire. M.R. declares no competing financial interests.

: The objective of this study was to assess the cost-effectiveness of pharmacokinetic-driven prophylaxis in severe haemophilia A patients. A microsimulation model was developed to evaluate the cost-effectiveness of pharmacokinetic-driven prophylaxis vs. standard prophylaxis and estimate cost, annual joint bleed rate (AJBR), and incremental cost-effectiveness ratio over a 1-year time horizon for a hypothetical population of 10 000 severe haemophilia A patients. A dose of 30 IU/kg per 48 h was assumed for standard prophylaxis. Pharmacokinetic prophylaxis was individually adjusted to maintain trough levels at least 1 and 5 IU/dl or less. AJBR was estimated on the relationship between factor VIII (FVIII) levels and bleeding rate reported in the literature. Sensitivity analyses were performed to assess the stability of the model and the reliability of results. The FVIII dose was reduced in the 27.8% of patients with a trough level more than 5 IU/dl on standard prophylaxis, with a negligible impact on AJBR (+0.1 bleed/year). The FVIII dose was increased in the 10.6% of patients with trough levels less than 1 IU/dl on standard prophylaxis, with a significant reduction of AJBR (-1.9 bleeds/year). On average, overall, pharmacokinetic-driven prophylaxis was shown to decrease the AJBR from 1.012 to 0.845 with a slight reduction of the infusion dose of 0.36 IU/kg, with total saving of 5 197&OV0556; per patient-year. Pharmacokinetic-driven prophylaxis was preferable (i.e. more effective and less costly) compared with standard prophylaxis, with savings of 31 205&OV0556; per bleed avoided. Pharmacokinetic-driven prophylaxis, accounting for patients' individual pharmacokinetic variability, appears to be a promising strategy to improve outcomes with efficient use of available resources in severe haemophilia A patients.

Alzheimer disease (AD) is a progressive neurodegenerative disorder leading to cognitive decline, neuropsychiatric symptoms, disability, caregiver burden, and premature death. It represents the most prevalent cause of dementia, and its incidence rates exponentially increase with increasing age. The number of Americans living with AD is rapidly increasing. An estimated 5.4 million Americans of all ages have AD in 2016. One in nine people aged 65 and older has AD, and by midcentury, someone in the United States will develop the disease every 33 sec. It is now accepted that neuroinflammation is a common feature of neurological disease. Inflammasomes, which are a multiprotein complex part of the innate immune system, induce inflammation in response to various stimuli, such as pathogens and stress. Inflammasomes activate proinflammatory caspases, such as caspase-1, leading to the activation of the proinflammatory cytokines interleukin (IL)-1b, IL-18, and IL-33, which promote neuroinflammation and brain pathologies. The nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing-3 (NLRP3) inflammasome is the best characterized in neurodegenerative diseases, in particular AD. Recent research suggests that NLRP3 could possibly be used in targeted therapies to alleviate neuroinflammation. Modulation of endogenous cellular defense mechanisms may be an innovative approach to therapeutic intervention in AD and other disorders associated with neuroinflammation and neurodegeneration. Herein, we introduce the hormetic dose-response concept and present possible mechanisms and applications to neuroprotection. We summarize the mechanisms involved in activation of the NLRP3 inflammasome and its role in neuroinflammation. We also address and propose the potential therapeutic utility of the nutritional antioxidants sulforaphane and hydroxytyrosol against particular signs and symptoms of AD. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Hemophilia is a rare bleeding disorder associated with spontaneous and post-traumatic bleeding. Each hemophilia patient requires a personalized approach to episodic or prophylactic treatment, but self-management can be challenging for patients, and avoidable bleeding may occur. Patient-tailored care may provide more effective prevention of bleeding, which in turn, may decrease the likelihood of arthropathy and associated chronic pain, missed time from school or work, and progressive loss of mobility. A strategy is presented here aiming to reduce or eliminate bleeding altogether through a holistic approach based on individual patient characteristics. In an environment of budget constraints, this approach would link procurement to patient outcome, adding incentives for all stakeholders to strive for optimal care and, ultimately, a bleed-free world.

Chronic inflammation has a key role in the pathogenesis of multiple diseases that represent major public health and financial concerns, including heart failure (HF), Alzheimer's disease (AD) and arthritis. Nuclear factor kappa β (NF-κβ) is a central component of inflammation; owing to its upstream signaling position, it is considered an attractive target for new anti-inflammatory therapeutics. Hydroxytyrosol is an orally bioavailable polyphenol, obtained from olives, which inhibits NF-κβ activity and has elicited promising efficacy signals in several inflammatory diseases. Here, we further examine the role of NF-κβ in inflammation, provide an introduction to natural products and their anti-inflammatory effects and explore the potential of hydroxytyrosol as a new approach to combating the burden of chronic inflammatory diseases., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Recombinant proteins are widely used today in many industries, including the biopharmaceutical industry, and can be expressed in bacteria, yeasts, mammalian and insect cell cultures, or in transgenic plants and animals. In addition, transgenic algae have also been shown to support recombinant protein expression, both from the nuclear and chloroplast genomes. However, to date, there are only a few reports on recombinant proteins expressed in the algal chloroplast. It is unclear whether this is because of few attempts or of limitations of the system that preclude expression of many proteins. Thus, we sought to assess the versatility of transgenic algae as a recombinant protein production platform. To do this, we tested whether the algal chloroplast could support the expression of a diverse set of current or potential human therapeutic proteins. Of the seven proteins chosen, >50% expressed at levels sufficient for commercial production. Three expressed at 2%-3% of total soluble protein, while a forth protein accumulated to similar levels when translationally fused to a well-expressed serum amyloid protein. All of the algal chloroplast-expressed proteins are soluble and showed biological activity comparable to that of the same proteins expressed using traditional production platforms. Thus, the success rate, expression levels, and bioactivity achieved demonstrate the utility of Chlamydomonas reinhardtii as a robust platform for human therapeutic protein production.

Several in vitro and in vivo studies have shown that olive phenols exert potent biological activities including, but not limited to, antioxidant actions. These activities are shared by phenols found in olives, olive oil, and olive mill wastewater (OMWW). The aim of this study was to investigate whether a commercially available OMWW preparation could influence some parameters of oxidative status in healthy human volunteers. Ninety-eight healthy subjects with normal body weight were recruited, and 5 mL of blood was drawn from their antecubital vein after an overnight fast of at least 12 h. After this, subjects were asked to ingest 2 mL of a commercially available OMWW preparation. Another 5 mL of blood was drawn 1 h after ingestion of the preparation. Plasma antioxidant capacity and total and reduced glutathione were measured. No difference in plasma antioxidant capacity was observed between baseline and 1 h after the ingestion of the extract. Conversely, a significant increase in total plasma glutathione concentration was measured. This increase involved both the reduced and oxidized forms of glutathione; hence, their ratio was unaffected by the treatment. The observed effects of OMWW on glutathione levels might be governed by the antioxidant response element (ARE)-mediated increase in phase II enzyme expression, including that of gamma-glutamylcysteine ligase and glutathione synthetase. Future studies on groups of individuals who may benefit from an increase in their glutathione levels, for example, the elderly, will further elucidate the biological activities of this formulation.

Look-through mutagenesis (LTM) is a multidimensional mutagenesis method that simultaneously assesses and optimizes combinatorial mutations of selected amino acids. The process focuses on a precise distribution within one or more complementarity determining region (CDR) domains and explores the synergistic contribution of amino acid side-chain chemistry. LTM was applied to an anti-TNF-alpha antibody, D2E7, which is a challenging test case, because D2E7 was highly optimized (K(d) = 1 nM) by others. We selected and incorporated nine amino acids, representative of the major chemical functionalities, individually at every position in each CDR and across all six CDRs (57 aa). Synthetic oligonucleotides, each introducing one amino acid mutation throughout the six CDRs, were pooled to generate segregated libraries containing single mutations in one, two, and/or three CDRs for each V(H) and V(L) domain. Corresponding antibody libraries were displayed on the cell surface of yeast. After positive binding selection, 38 substitutions in 21 CDR positions were identified that resulted in higher affinity binding to TNF-alpha. These beneficial mutations in both V(H) and V(L) were represented in two combinatorial beneficial mutagenesis libraries and selected by FACS to produce a convergence of variants that exhibit between 500- and 870-fold higher affinities. Importantly, these enhanced affinities translate to a 15- to 30-fold improvement in in vitro TNF-alpha neutralization in an L929 bioassay. Thus, this LTM/combinatorial beneficial mutagenesis strategy generates a comprehensive energetic map of the antibody-binding site in a facile and rapid manner and should be broadly applicable to the affinity maturation of antibodies and other proteins.

Fruit and vegetable simple and polyphenols are potent antioxidants. One of the most effective in terms of free radical scavenging is 3,4-dihydroxyphenyl ethanol or hydroxytyrosol (HT), a simple phenol found predominantly in Olea europea, or the olive plant. HT is most abundant in the aqueous fraction of olive pulp with trace amounts in the olive oil fraction and in the leaves. For these experiments, we evaluated the anti-inflammatory activity of olive vegetation water (OVW), which we showed previously to have potent antioxidant activity. Because some simple phenols and polyphenols with antioxidant activity have shown varying anti-inflammatory activities, we tested OVW and HT for their ability to inhibit the production of tumor necrosis factor-alpha (TNF-alpha), a pivotal cytokine in inflammation. In lipopolysaccharide (LPS)-treated BALB/c mice, a model system of inflammation, OVW at a dose of 125 mg/mouse (500 mg/kg) reduced serum TNF-alpha levels by 95%. In the human monocyte cell line, THP-1, OVW reduced LPS-induced TNF-alpha production by 50% at a concentration of 0.5 g/L (equivalent to approximately 0.03 g/L simple and polyphenols). OVW had no toxic effects in vitro or in vivo. When OVW was combined with glucosamine, a component of proteoglycans and glycoproteins that was shown to decrease inducible nitric oxide synthase production in cultured macrophage cells, the 2 compounds acted synergistically to reduce serum TNF-alpha levels in LPS-treated mice. These findings suggest that a combination of OVW and glucosamine may be an effective therapy for a variety of inflammatory processes, including rheumatoid and osteoarthritis.

The toxicity profile of HIDROX (Hydrolyzed Aqueous Olive Pulp Extract; OPE) was characterized in a series of toxicology studies. A limit dosage of 2000 mg/kg produced no toxicity in mice (acute oral NOAEL: 2000 mg/kg). In rats, an acute oral NOAEL of 2000 mg/kg was established, based on reductions in weight gains in both sexes at 5000 mg/kg. Reduced gains in female rats at 1500 and 2000 mg/kg were not significantly different from control values. Daily oral dosages of 1000, 1500 and 2000 mg/kg/day for 90 days produced small decreases in body weight gains at 2000 mg/kg/day in the male rats and in all groups of female rats. Feed consumption was comparable to controls. There were no adverse clinical, hematologic, biochemical, organ weight or gross necropsy effects. Focal, minimal or mild hyperplasia of the mucosal squamous epithelium of the limiting ridge of the forestomach occurred in some rats at 2000 mg/kg/day; this change was attributed to local irritation by repeated intubation of large volumes of viscous, granular dosing suspension. A NOAEL of 2000 mg/kg/day was established for the 90-day study, based on the lack of significant adverse effects. Toxicokinetic data indicated that hydroxytyrosol (HT, the major component of OPE) was rapidly absorbed. Mean concentrations were measurable through 1 to 4 hours (t(last)) at 1000 and 1500 mg/kg/day and through 8 hours at 2000 mg/kg/day. Dosages of OPE ranging from 500 to 2000 mg/kg/day did not adversely affect any of the mating, fertility, delivery or litter parameters investigated in an oral rat dosage-range reproduction study. Adverse effects were also absent in a rat developmental toxicity study in which pregnant dams were treated with 1000, 1500 or 2000 mg/kg/day on days 6 through 20 of gestation. Plasma levels for pregnant and lactating rats were comparable to non-pregnant rats; minimal levels crossed the placenta. Quantifiable levels were not identified in maternal milk or plasma from nursing pups. A bacterial reverse mutation and a CHO chromosome aberration assay revealed evidence of mutagenic activity at high dosages with S9 metabolic activation. However, three rat micronucleus evaluations performed after single and repeated (28-day) dosages of up to 2000 mg/kg/day and dosages of 5000 mg/kg/day for 29 days resulted in negative findings; therefore, OPE was not considered to be mutagenic in this in vivo assay.

A solid-phase approach using trinucleotide building blocks and functionalized cellulose as solid support was used to synthesize the 22 oligodeoxynucleotides that constitute a totally synthetic gene coding for a 43 amino acid peptide. Gastric inhibitory Polypeptide. The overall strategy used for the design and synthesis of the oligomers is described.

Thymosin alpha 1, an immune restorative polypeptide hormone, was synthesized in Escherichia coli by using recombinant DNA cloning techniques. Based on the known amino acid sequence, a gene coding for the thymosin alpha 1 polypeptide chain was designed and enzymatically assembled from chemically synthesized oligodeoxyribonucleotide fragments. The gene was ligated into plasmid pBR322 and placed under lac operon control, and N alpha-desacetylthymosin alpha 1 was expressed as part of a beta-galactosidase chimeric protein. Cyanogen bromide cleavage of this protein gave a mixture of polypeptides, among which thymosin alpha 1 activity was detected by radioimmunoassay (RIA). The E. coli product is identical with native thymosin alpha 1 isolated from calf thymus in the amino acid sequence but lacks the N-terminal acetyl group. Results of a guinea pig migration inhibition factor (MIF) assay, a terminal deoxyribonucleotidyl transferase (TdT) assay, and radioimmunoassay indicate that the N alpha-desacetylthymosin alpha 1 produced by deoxyribonucleic acid (DNA) cloning techniques has biological activity equivalent to that of the native hormone.

A multipurpose cloning site has been introduced into the gene for beta-galactosidase (beta-D-galactosidegalactohydrolase, EC 3.21.23) on the single-stranded DNA phage M13mp2 (Gronenborn, B. and Messing, J., (1978) Nature 272, 375-377) with the use of synthetic DNA. The site contributes 14 additional codons and does not affect the ability of the lac gene product to undergo intracistronic complementation. Two restriction endonuclease cleavage sites in the viral gene II were removed by single base-pair mutations. Using the new phage M13mp7, DNA fragments generated by cleavage with a variety of different restriction endonucleases can be cloned directly. The nucleotide sequences of the cloned DNAs can be determined rapidly by DNA synthesis using chain terminators and a synthetic oligonucleotide primer complementary to 15 bases preceeding the new array of restriction sites.

A human leukocyte interferon cDNA was enzymatically synthesized, inserted into the vector pBR322, and cloned in Escherichia coli. The DNA sequence codes for a 23-amino acid signal peptide followed by an interferon polypeptide of 165 amino acids. An expression plasmid was constructed which permits the synthesis in E. coli of 2.5 x 10(8) units of interferon per litre of culture. This LeIF protected squirrel monkeys from lethal encephalomyocarditis virus infection.

A gene for somatostatin, a mammalian peptide (14 amino acid residues) hormone, was synthesized by chemical methods. This gene was fused to the Escherichia coli beta-galactosidase gene on the plasmid pBR322. Transformation of E. coli with the chimeric plasmid DNA led to the synthesis of a polypeptide including the sequence of amino acids corresponding to somatostatin. In vitro, active somatostatin was specifically cleaved from the large chimeric protein by treatment with cyanogen bromide. This represents the first synthesis of a functional polypeptide product from a gene of chemically synthesized origin.

A gene coding for a calmodulin was synthesized and expressed in Escherichia coli. The gene was produced by the enzymatic ligation of 61 chemically synthesized DNA fragments. The gene possesses 27 unique, regularly spaced, restriction endonuclease cleavage sites to facilitate gene mutagenesis by the replacement of specific gene segments with synthetic double-stranded DNA. An expression vector containing the calmodulin gene was used to transform E. coli. Purification and characterization of calmodulin (VU-1 calmodulin) expressed by these transformants showed that it lacks two posttranslational modifications: an amino-terminal blocking group and N epsilon, N epsilon, N epsilon-trimethyllysine at position 115. The cyclic nucleotide phosphodiesterase activator properties of VU-1, higher plant, and vertebrate calmodulins were not statistically different. However, VU-1 calmodulin was found to activate nicotinamide adenine dinucleotide (NAD) kinase to a maximal level that was at least 3-fold higher than that found with higher plant and vertebrate calmodulins. This higher level of activation is also characteristic of calmodulins from Dictyostelium discoideum and Chlamydomonas reinhardtii [Roberts, D. M., Burgess, W. H., & Watterson, D. M. (1984) Plant Physiol. 75, 796-798; Marshak, D. R., Clarke, M., Roberts, D. M., & Watterson, D. M. (1984) Biochemistry 23, 2891-2899]. The only common feature among Dictyostelium, Chlamydomonas, and VU-1 calmodulins not found in higher plant and vertebrate calmodulins is an unmethylated lysine at position 115. The results indicate that the lack of methylation of lysine-115 may contribute to the maximal level of NAD kinase activation.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA coding for human growth hormone was constructed by using chemically synthesised DNA in conjunction with enzymatically prepared cDNA. This 'hybrid' gene was expressed in Escherichia coli under the control of the lac promoter. A polypeptide was produced having the size and immunological properties characteristic of mature human growth hormone.

We have explored analogs of 2'-5'-linked andeylic acid trimer (2-5A): 3'-O-methylated 2-5A, 2'-end modified adenylate trimer with deoxyadenosine or araadenine, methyl phosphonate and methyl phosphorotriester analogs as potential antiviral agents. For the treatment of virus infections, 2-5A and its analogs may serve in lieu of interferon, however, the use of 2-5A has two serious limitations: it is presumed to be impermeable to most cells, and moreover, cellular enzymes rapidly degrade it. Methylated analogs of 2-5A core strongly inhibited virus growth when added directly to cells in culture. 2'-End modified adenylate trimer with araadenine also inhibited virus growth, however, neither 2-5A nor other analogs showed any significant antiviral activity. The inhibition of virus growth was not due to the toxic effect of these compounds on cell growth as they had no inhibitory effect on the growth of uninfected cells.

The synthesis of oligothymidilic acids, (dT)m (where m = 4, 7, 10, 13, 16, 19, 22, and 25), was carried out using a solid phase approach in combination with the modified phosphotriester methodology developed in solution. Cellulose was used as the solid support after its functionalization with a specially featured dinucleoside diphosphate, 5'-0-p-chlorophenylphospho-2'(3')-0-acetyluridilyl-[2'(3')-3']-5'-0-dimethoxytritylthymidine p-chlorophenylester. The fully protected trideoxynucleoside triphosphate containing only thymidine was repeatedly used to elongate the oligonucleotide chain in the 3'-5' direction. Individual coupling yields ranged from 45% to 75%. The total time needed to prepare (dT)25 was four days. Similarly, the tridecanucleotide d(AGAAGGTACTTTT) was synthesized in good yield. The results show that this approach can be used for a fast and economic way to synthesize oligodeoxynucleotides.

The conformation of d (CG)n oligomers with n = 2,3 has been studied in aqueous solution in the presence of high salt concentration. A minimum n value of three is necessary to obtain a left-handed Z-helix. When d (CG)3 is flanked by three non Z-helicogenic alternating AT sequences the left-handed helix is unstable and a B-type conformation is obtained also at high salt concentration.

A gene has been constructed which codes for an analog of human proinsulin in which the normal 35-amino acid connecting peptide is replaced by a "mini-C" peptide of six amino acids (Arg-Arg-Gly-Ser-Lys-Arg). The gene, composed of oligonucleotide fragments synthesized by the triester method, was cloned and expressed as a beta-galactosidase hybrid protein. The proinsulin analog was separated from beta-galactosidase by cyanogen bromide cleavage and purified. Controlled disulfide exchange in the S-sulfonate of the analog generated a molecule having high-pressure liquid chromatography (HPLC) and radioimmunoassay (RIA) behavior consistent with a proinsulin-like structure.

We have identified the startpoint for transcription in vitro of the tetracycline resistance gene (tet) of pBR322 and several deletion and insertion mutations which alter tet promoter structure. Tetracycline resistance in host bacteria correlates qualitatively with the efficiency of DNA fragments from these plasmids to promote tet transcription in vitro. Only in active promoters could we find by computer analysis promoter structures in which the -10 and -35 sequences and the relative spacing of the two regions agree with consensus sequence determinants. These data support the current model of the E. coli promoter sequence. Two promoter mutants gave heterogeneous 5' termini with additional A residues not encoded by the DNA sequence.

A cDNA library was constructed using mRNA from human fibroblasts induced with poly(I):poly(C). A bacterial clone containing fibroblast interferon cDNA sequences was identified by hybridization to a cDNA probe synthesized using deoxyoligonucleotide primers which hybridize to fibroblast interferon mRNA specifically. Expression plasmids were constructed which permitted the synthesis in E. coli of 8 x 10(7) units of human fibroblast interferon per liter of culture. The bacterially produced fibroblast interferon is indistinguishable from authentic human fibroblast interferon by several criteria.

The conformation of d(TA)3 (CG)3 has been studied in aqueous solution in the presence of high salt concentration. The oligonucleotide forms in solution concatamers. In the presence of 5 M NaC1 the concatamer is built up of d(CG)3 blocks in Z-conformation and of d(TA)3 blocks in B-conformation. In the presence of 4 M LiCl both blocks in the concatamer display a B-type structure.