Early High-Titer Plasma Therapy to Prevent Severe Covid-19 in Older Adults.

BACKGROUND: Therapies to interrupt the progression of early coronavirus disease 2019 (Covid-19) remain elusive. Among them, convalescent plasma administered to hospitalized patients has been unsuccessful, perhaps because antibodies should be administered earlier in the course of illness. METHODS: We conducted a randomized, double-blind, placebo-controlled trial of convalescent plasma with high IgG titers against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in older adult patients within 72 hours after the onset of mild Covid-19 symptoms. The primary end point was severe respiratory disease, defined as a respiratory rate of 30 breaths per minute or more, an oxygen saturation of less than 93% while the patient was breathing ambient air, or both. The trial was stopped early at 76% of its projected sample size because cases of Covid-19 in the trial region decreased considerably and steady enrollment of trial patients became virtually impossible. RESULTS: A total of 160 patients underwent randomization. In the intention-to-treat population, severe respiratory disease developed in 13 of 80 patients (16%) who received convalescent plasma and 25 of 80 patients (31%) who received placebo (relative risk, 0.52; 95% confidence interval [CI], 0.29 to 0.94; P = 0.03), with a relative risk reduction of 48%. A modified intention-to-treat analysis that excluded 6 patients who had a primary end-point event before infusion of convalescent plasma or placebo showed a larger effect size (relative risk, 0.40; 95% CI, 0.20 to 0.81). No solicited adverse events were observed. CONCLUSIONS: Early administration of high-titer convalescent plasma against SARS-CoV-2 to mildly ill infected older adults reduced the progression of Covid-19. (Funded by the Bill and Melinda Gates Foundation and the Fundación INFANT Pandemic Fund; Dirección de Sangre y Medicina Transfusional del Ministerio de Salud number, PAEPCC19, Plataforma de Registro Informatizado de Investigaciones en Salud number, 1421, and ClinicalTrials.gov number, NCT04479163.).

Prospective Latin American cohort evaluating outcomes of patients with COVID-19 and abnormal liver tests on admission.

INTRODUCTION & OBJECTIVES: The independent effect of liver biochemistries as a prognostic factor in patients with COVID-19 has not been completely addressed. We aimed to evaluate the prognostic value of abnormal liver tests on admission of hospitalized patients with COVID-19. MATERIALS & METHODS: We performed a prospective cohort study including 1611 hospitalized patients with confirmed SARS-CoV-2 infection from April 15, 2020 through July 31, 2020 in 38 different Hospitals from 11 Latin American countries. We registered clinical and laboratory parameters, including liver function tests, on admission and during hospitalization. All patients were followed until discharge or death. We fit multivariable logistic regression models, further post-estimation effect through margins and inverse probability weighting. RESULTS: Overall, 57.8% of the patients were male with a mean age of 52.3 years, 8.5% had chronic liver disease and 3.4% had cirrhosis. Abnormal liver tests on admission were present on 45.2% (CI 42.7-47.7) of the cohort (n = 726). Overall, 15.1% (CI 13.4-16.9) of patients died (n = 244). Patients with abnormal liver tests on admission presented higher mortality 18.7% (CI 15.9-21.7), compared to those with normal liver biochemistries 12.2% (CI 10.1-14.6); P < .0001). After excluding patients with history of chronic liver disease, abnormal liver tests on admission were independently associated with death [OR 1.5 (CI 1.1-2.0); P = 0.01], and severe COVID-19 (2.6 [2.0-3.3], P < .0001), both adjusted by age, gender, diabetes, pneumonia and body mass index >30. CONCLUSIONS: The presence of abnormal liver tests on admission is independently associated with mortality and severe COVID-19 in hospitalized patients with COVID-19 infection and may be used as surrogate marker of inflammation. CLINICALTRIALS.GOV: NCT04358380.

Comparison of different prognostic scores for patients with cirrhosis hospitalized with SARS-CoV-2 infection.

INTRODUCTION AND OBJECTIVES: Viral infections have been described to increase the risk of decompensation in patients with cirrhosis. We aimed to determine the effect of SARS-CoV-2 infection on outcome of hospitalized patients with cirrhosis and to compare the performance of different prognostic models for predicting mortality. PATIENTS: We performed a prospective cohort study including 2211 hospitalized patients with confirmed SARS-CoV-2 infection from April 15, 2020 through October 1, 2020 in 38 Hospitals from 11 Latin American countries. We registered clinical and laboratory parameters of patients with and without cirrhosis. All patients were followed until discharge or death. We evaluated the prognostic performance of different scoring systems to predict mortality in patients with cirrhosis using ROC curves. RESULTS: Overall, 4.6% (CI 3.7-5.6) subjects had cirrhosis (n = 96). Baseline Child-Turcotte-Pugh (CTP) class was assessed: CTP-A (23%), CTP-B (45%) and CTP-C (32%); median MELD-Na score was 19 (IQR 14-25). Mortality was 47% in patients with cirrhosis and 16% in patients without cirrhosis (P < .0001). Cirrhosis was independently associated with death [OR 3.1 (CI 1.9-4.8); P < .0001], adjusted by age, gender, and body mass index >30. The areas under the ROC curves for performance evaluation in predicting 28-days mortality for Chronic Liver Failure Consortium (CLIF-C), North American Consortium for the Study of End-Stage Liver Disease (NACSELD), CTP score and MELD-Na were 0.85, 0.75, 0.69, 0.67; respectively (P < .0001). CONCLUSIONS: SARS-CoV-2 infection is associated with elevated mortality in patients with cirrhosis. CLIF-C had better performance in predicting mortality than NACSELD, CTP and MELD-Na in patients with cirrhosis and SARS-CoV-2 infection. Clinicaltrials.gov:NCT04358380.

Anti-Neuroblastoma Properties of a Recombinant Sunflower Lectin.

According to their sugar recognition specificity, plant lectins are proposed as bioactive proteins with potential in cancer treatment and diagnosis. Helja is a mannose-specific jacalin-like lectin from sunflower which was shown to inhibit the growth of certain fungi. Here, we report its recombinant expression in a prokaryotic system and its activity in neurobalstoma cells. Helja coding sequence was fused to the pET-32 EK/LIC, the enterokinase/Ligation-independent cloning vector and a 35 kDa protein was obtained in Escherichia coli representing Helja coupled to thioredoxin (Trx). The identity of this protein was verified using anti-Helja antibodies. This chimera, named Trx-rHelja, was enriched in the soluble bacterial extracts and was purified using Ni+2-Sepharose and d-mannose-agarose chromatography. Trx-rHelja and the enterokinase-released recombinant Helja (rHelja) both displayed toxicity on human SH-SY5Y neuroblastomas. rHelja decreased the viability of these tumor cells by 75% according to the tetrazolium reduction assay, and microscopic analyses revealed that the cell morphology was disturbed. Thus, the stellate cells of the monolayer became spheroids and were isolated. Our results indicate that rHelja is a promising tool for the development of diagnostic or therapeutic methods for neuroblastoma cells, the most common solid tumors in childhood.

Metabolite profiling and cytotoxic activity of Andean potatoes: Polyamines and glycoalkaloids as potential anticancer agents in human neuroblastoma cells in vitro.

Andean potatoes (Solanum tuberosum L. ssp. andigena) are a good source of dietary antioxidant polyphenols. We have previously demonstrated that polyphenol extracts from Andean potato tubers exerted a dose-dependent cytotoxic effect in human neuroblastoma SH-SY5Y cells, being skin extracts more potent than flesh ones. In order to gain insight into the bioactivities of potato phenolics, we investigated the composition and the in vitro cytotoxic activity of total extracts and fractions of skin and flesh tubers of three Andean potato cultivars (Santa María, Waicha, and Moradita). Potato total extracts were subjected to liquid-liquid fractionation using ethyl acetate solvent in organic and aqueous fractions. We analyzed both fractions by HPLC-DAD, HPLC-ESI-MS/MS, and HPLC-HRMS. Results corroborated the expected composition of each fraction. Organic fractions were rich in hydroxycinnamic acids (principally chlorogenic acid isomers), whereas aqueous fractions contained mainly polyamines conjugated with phenolic acids, glycoalkaloids, and flavonoids. Aqueous fractions were cytotoxic against SH-SY5Y cells and even more potent than their respective total extracts. Treatment with a combination of both fractions showed a similar cytotoxic response to the corresponding extract. According to correlation studies, it is tempting to speculate that polyamines and glycoalkaloids are crucial in inducing cell death. Our findings indicate that the activity of Andean potato extracts is a combination of various compounds and contribute to the revalorization of potato as a functional food.

The Water-Soluble Chitosan Derivative, N-Methylene Phosphonic Chitosan, Is an Effective Fungicide against the Phytopathogen Fusarium eumartii.

Chitosan has been considered an environmental-friendly polymer. However, its use in agriculture has not been extended yet due to its relatively low solubility in water. N-Methylene phosphonic chitosan (NMPC) is a water-soluble derivative prepared by adding a phosphonic group to chitosan. This study demonstrates that NMPC has a fungicidal effect on the phytopathogenic fungus Fusarium solani f. sp. eumartii (F. eumartii) judged by the inhibition of F. eumartti mycelial growth and spore germination. NMPC affected fungal membrane permeability, reactive oxygen species production, and cell death. Also, this chitosan-derivative exerted antifungal effects against two other phytopathogens, Botrytis cinerea, and Phytophthora infestans. NMPC did not affect tomato cell viability at the same doses applied to these phytopathogens to exert fungicide action. In addition to water solubility, the selective biological cytotoxicity of NMPC adds value in its application as an antimicrobial agent in agriculture.

Brain cholinergic impairment in liver failure.

The cholinergic system is involved in specific behavioural responses and cognitive processes. Here, we examined potential alterations in the brain levels of key cholinergic enzymes in cirrhotic patients and animal models with liver failure. An increase (~30%) in the activity of the acetylcholine-hydrolyzing enzyme, acetylcholinesterase (AChE) is observed in the brain cortex from patients deceased from hepatic coma, while the activity of the acetylcholine-synthesizing enzyme, choline acetyltransferase, remains unaffected. In agreement with the human data, AChE activity in brain cortical extracts of bile duct ligated (BDL) rats was increased (~20%) compared to controls. A hyperammonemic diet did not result in any further increase of AChE levels in the BDL model, and no change was observed in hyperammonemic diet rats without liver disease. Portacaval shunted rats which display increased levels of cerebral ammonia did not show any brain cholinergic abnormalities, confirming that high ammonia levels do not play a role in brain AChE changes. A selective increase of tetrameric AChE, the major AChE species involved in hydrolysis of acetylcholine in the brain, was detected in both cirrhotic humans and BDL rats. Histological examination of BDL and non-ligated rat brains shows that the subcellular localization of both AChE and choline acetyltransferase, and thus the accessibility to their substrates, appears unaltered by the pathological condition. The BDL-induced increase in AChE activity was not parallelled by an increase in mRNA levels. Increased AChE in BDL cirrhotic rats leads to a pronounced decrease (~50-60%) in the levels of acetylcholine. Finally, we demonstrate that the AChE inhibitor rivastigmine is able to improve memory deficits in BDL rats. One week treatment with rivastigmine (0.6 mg/kg; once a day, orally, for a week) resulted in a 25% of inhibition in the enzymatic activity of AChE with no change in protein composition, as assessed by sucrose density gradient fractionation and western blotting analysis. In conclusion, this study is the first direct evidence of a cholinergic imbalance in the brain as a consequence of liver failure and points to the possible role of the cholinergic system in the pathogenesis of hepatic encephalopathy.

Association between acetylcholinesterase and beta-amyloid peptide in Alzheimer's cerebrospinal fluid.

The altered expression of acetylcholinesterase (AChE) in the brains of patients with Alzheimer's disease (AD) has raised much interest of late. Despite an overall decrease in the AD brain, the activity of AChE increases around beta-amyloid plaques and indeed, the beta-amyloid peptide (Abeta) can influence AChE levels. Such evidence stimulated our interest in the possibility that the levels of AChE and amyloid might vary together in AD. We previously found that the different AChE forms present in both the brain and in the cerebrospinal fluid (CSF) of AD patients varied in conjunction with abnormal glycosylation. Thus, the alterations in glycosylation are correlated with the accumulation of a minor subspecies of AChE monomers. We also recently analysed whether long-term exposure to the cholinesterase inhibitor (ChE-I) donepezil influences the AChE species found in AD CSF. The marked increase in CSF-AChE activity in AD patients following long-term treatment with donepezil was not paralleled by a rise in this subset of light variants. Hence, the correlation with the levels of CSF-Abeta is unique to these AChE species in patients receiving such treatment. The aim of this report is to review the links between AChE and beta-amyloid, and to discuss the significance of the responses of the distinct AChE species to ChE-I during the treatment of AD.

Bactericidal and Cytotoxic Activities of Polyphenol Extracts from Solanum tuberosum spp. tuberosum and spp. andigena Cultivars on Escherichia coli and Human Neuroblastoma SH-SY5Y Cells In Vitro.

Potatoes (Solanum tuberosum L.) are a good source of dietary antioxidant polyphenols. This study investigated the potential antioxidant, bactericidal, and cytotoxic activities in vitro of the phenolic compounds present in tubers of one S. tuberosum spp. tuberosum (Summerside), and three S. tuberosum spp. andigena (landraces Moradita, Waicha, and Santa María) cultivars. Both the content of phenolic acids, chlorogenic acids (CGAs) being the most abundant, and the antioxidant activity were higher in extracts from skin than from flesh. Extracts from Moradita flesh and Summerside skin showed bactericidal activity against Escherichia coli ATCC 25922 but failed to inhibit pathogenic E. coli O157. Both extracts lack pigmentation but do contain 5-CGA, caffeic, and ferulic acids. Positive control with gentamicin and commercial 5-CGA resulted in a complete inhibition of bacterial growth. In addition, all potato extracts and commercial 5-CGA diminished dose-dependently human neuroblastoma SH-SY5Y cell viability. Skin extracts were more potent than flesh extracts. Among flesh extracts, Moradita was the most potent. Together, our results suggest that Moradita flesh could provide a desirable source of important health-promoting substances. Findings indicate that the biological activity of potato extracts is a combination of various bioactive compounds and contribute to the revalorization of potato as a functional food.

Altered glycosylation of acetylcholinesterase in the Creutzfeldt-Jakob cerebrospinal fluid.

Several neurodegenerative disorders present deficiencies in the cholinergic system. Scarce research on prion encephalopathies has examined the levels of cholinergic pathway-related enzymes. Acetylcholinesterase (AChE) is expressed as several molecular forms. The potential importance of these variants is increased by the possibility that AChE has roles other than acetylcholine hydrolysis. We investigated the levels of AChE, its molecular forms, and glycosylation in the cerebrospinal fluid (CSF) from Creutzfeldt-Jakob disease (CJD) patients.

Altered expression of brain acetylcholinesterase in FTDP-17 human tau transgenic mice.

Pathological hyperphosphorylation and aggregation of the tau protein is associated with dementia and can be the central cause of neurodegeneration. Here, we examined potential alterations in the level of the cholinergic enzyme acetylcholinesterase (AChE) in the brain of transgenic mice (Tg-VLW) expressing human tau mutations. Overexpression of mutant hyperphosphorylated tau (P-tau) led to an increase in the activity of AChE in the brain of Tg-VLW mice, paralleled by an increase in AChE protein and transcripts; whereas the levels of the enzyme choline acetyltransferase remained unaffected. VLW tau overexpression in SH-SY5Y cells also increased AChE activity levels. All major molecular forms of AChE were increased in the Tg-VLW mice, including tetrameric AChE, which is the major species involved in hydrolysis of acetylcholine in the brain. Colocalization of human P-tau and AChE supports the conclusion that P-tau can act to increase AChE. This study is the first direct evidence of a modulatory effect of P-tau on brain AChE expression.

Changes in acetylcholinesterase expression are associated with altered presenilin-1 levels.

We have previously identified presenilin-1 (PS1), the active component of the γ-secretase complex, as an interacting protein of the amyloid-associated enzyme acetylcholinesterase (AChE). In this study, we have explored the consequences of AChE-PS1 interactions. Treatment of SH-SY5Y cells with the AChE-inhibitor tacrine decreased PS1 levels, in parallel with increase in the secretion of amyloid precursor protein APPα, whereas the cholinergic agonist carbachol had no effect on PS1. AChE knockdown with siRNA also decreased PS1 levels, while AChE overexpression exerted opposing effect. AChE-deficient also had decreased PS1. Mice administered with tacrine or donepezil displayed lower levels of brain PS1. However, sustained AChE inhibition failed to exert long-term effect on PS1. This limited duration of response may be due to AChE upregulation caused by chronic inhibition. Finally, we exposed SH-SY5Y cells to ß-amyloid (Aß)42 which triggered elevation of both AChE and PS1 levels. The Aß42-induced PS1 increase was abolished by siRNA AChE pretreatment, suggesting that AChE may participate in the pathological feedback loop between PS1 and Aß. Our results provide insight into AChE-amyloid interrelationships.

Presenilin 1 interacts with acetylcholinesterase and alters its enzymatic activity and glycosylation.

Presenilin 1 (PS1) plays a critical role in the gamma-secretase processing of the amyloid precursor protein to generate the beta-amyloid peptide, which accumulates in plaques in the pathogenesis of Alzheimer's disease (AD). Mutations in PS1 cause early onset AD, and proteins that interact with PS1 are of major functional importance. We report here the coimmunoprecipitation of PS1 and acetylcholinesterase (AChE), an enzyme associated with amyloid plaques. Binding occurs through PS1 N-terminal fragment independent of the peripheral binding site of AChE. Subcellular colocalization of PS1 and AChE in cultured cells and coexpression patterns of PS1 and AChE in brain sections from controls and subjects with sporadic or familial AD indicated that PS1 and AChE are located in the same intracellular compartments, including the perinuclear compartments. A PS1-A246E pathogenic mutation expressed in transgenic mice leads to decreased AChE activity and alteration of AChE glycosylation and the peripheral binding site, which may reflect a shift in protein conformation and disturbed AChE maturation. In both the transgenic mice and humans, mutant PS1 impairs coimmunoprecipitation with AChE. The results indicate that PS1 can interact with AChE and influence its expression, supporting the notion of cholinergic-amyloid interrelationships.

Gene cloning and heterologous synthesis of a haloalkaliphilic extracellular protease of Natrialba magadii (Nep).

The gene encoding the protease Nep secreted by the haloalkaliphilic archaeon Natrialba magadii was cloned and sequenced. Upstream of the nep gene, a region related to haloarchaeal TATA-box and BRE-like consensus sequences was identified. The nep-encoded polypeptide had a molecular mass of 56.4 kDa, a pI of 3.77 and included a 121-amino acid propeptide not present in the mature Nep. A Tat motif (GRRSVL) was also identified at residues 10-15 suggesting it is a substrate of the Tat pathway. The primary sequence of Nep was closely related to serine proteases of the subtilisin family from archaea and bacteria (50-85% similarity). The nep gene was expressed in Escherichia coli and Haloferax volcanii resulting in production of active Nep protease. In contrast to the recombinant E. coli strains in which Nep activity was only detected in cell lysate, high levels of Nep protein and activity were detected in the culture medium of stationary phase recombinant Hfx. volcanii strains. The Hfx. volcanii synthesized protease was active in high salt, high pH and high DMSO. This study provides the first molecular characterization of a halolysin-like protease from alkaliphilic haloarchaea and is the first description of a recombinant system that facilitates high-level secretion of a haloarchaeal protease.

Cerebrospinal fluid acetylcholinesterase changes after treatment with donepezil in patients with Alzheimer's disease.

We analyzed whether donepezil differently influences acetylcholinesterase (AChE) variants from cerebrospinal fluid (CSF) in patients with Alzheimer's disease (AD) after long-term treatment. Overall CSF-AChE activity in AD patients before treatment was not different from controls, but the ratio between the major tetrameric form, G(4), and the smaller G(1) and G(2) species was significantly lower. AChE levels at study outset were found to correlate positively with beta-amyloid (1-42) (Abeta42). When patients were re-examined after 12 months treatment with donepezil, there was a remarkable increase in both the G(4) and the lighter species of CSF AChE. As compared with placebo, donepezil caused decreases in the percentage of AChE that failed to bind to the lectin concanavalin A and the antibody AE1. These non-binding species comprised primarily a small subset of G(1) and G(2) forms. In treated patients, these light variants were the only subset of CSF AChE that correlated with CSF-Abeta42 levels. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that a 77-kDa band, attributed in part to inactive AChE, was lower in AD patients than in controls. Unlike enzyme activity, the intensity of this band did not increase after donepezil treatment. The varying responses of different AChE species to ChE-I treatment suggest different modes of regulation, which may have therapeutic implications.

Altered glycosylation of acetylcholinesterase in Creutzfeldt-Jakob disease.

Changes in the glycosylation pattern of brain proteins have been associated with Creutzfeldt-Jakob disease (CJD). We have investigated the glycosylation status of acetylcholinesterase (AChE) by lectin binding assay. Our data show that in lumbar CSF from definite and probable sporadic CJD cases AChE activity is lower compared with that in age-matched controls. We also show, for the first time, that AChE glycosylation is altered in CJD CSF and brain. Unlike Alzheimer's disease, in which an alteration in both the glycosylation and levels of AChE molecular forms is observed, the abnormal glycosylation of AChE in CJD appears to be unrelated to changes in molecular forms of this enzyme. These findings suggest that altered AChE glycosylation in CJD may be a consequence of the general perturbation of the glycosylation machinery that affects prion protein, as well as other proteins. The diagnostic potential of these changes remains to be explored.