Acanthamoeba castellanii (genotype T4) stimulates the production of interleukin-10 as well as pro-inflammatory cytokines in THP-1 cells, human peripheral blood mononuclear cells and human monocyte-derived macrophages

Free-living amoebae of the genus Acanthamoeba can cause severe and chronic infections in humans, mainly localized in immune privileged sites, as the brain and the eye. Monocytes/macrophages are thought to be involved in Acanthamoeba infections, but little is known about how these facultative parasites influence their functions. The aim of this work was to investigate the effects of Acanthamoeba on human monocytes/macrophages, during the early phase of infection. Herein, THP-1 cells, primary human monocytes isolated from peripheral blood and human monocyte-derived macrophages were either co-incubated with trophozoites of a clinical isolate of Acanthamoeba (genotype T4) or stimulated with amoeba-derived cell free conditioned medium. Production of pro-inflammatory cytokines (TNF-α, IL-6, IL-12), anti-inflammatory cytokine (IL-10) and chemokine (IL-8) was evaluated at specific hours post-stimulation (ranging from 1.30 h to 23 h). We showed that both Acanthamoeba trophozoites and soluble amoebic products induce an early anti-inflammatory monocyte-macrophage phenotype, characterized by a significant production of IL-10; furthermore, challenge with either trophozoites or their soluble metabolites stimulate both pro-inflammatory cytokines and chemokine production, suggesting that this protozoan infection may result from the early induction of coexisting, opposed immune responses. Results reported in this paper confirm that the production of pro-inflammatory cytokines and chemokines by monocytes and macrophages can play a role in the development of the inflammatory response during Acanthamoeba infections. Furthermore, we demonstrate for the first time that Acanthamoeba stimulates IL-10 production in human innate immune cells, which might both promote the immune evasion of Acanthamoeba and limit the induced inflammatory response

Metagenomics and microscope revealed T. trichiura and other intestinal parasites in a cesspit of an Italian nineteenth century aristocratic palace

This study evidenced the presence of parasites in a cesspit of an aristocratic palace of nineteenth century in Sardinia (Italy) by the use of classical paleoparasitological techniques coupled with next-generation sequencing. Parasite eggs identified by microscopy included helminth genera pathogenic for humans and animals: the whipworm Trichuris sp., the roundworm Ascaris sp., the flatworm Dicrocoelium sp. and the fish tapeworm Diphyllobothrium sp. In addition, 18S rRNA metabarcoding and metagenomic sequencing analysis allowed the first description in Sardinia of aDNA of the human specific T. trichiura species and Ascaris genus. Their presence is important for understanding the health conditions, hygiene habits, agricultural practices and the diet of the local inhabitants in the period under study

Colorectal Cancer Stage at Diagnosis Before vs During the COVID-19 Pandemic in Italy

IMPORTANCE Delays in screening programs and the reluctance of patients to seek medical attention because of the outbreak of SARS-CoV-2 could be associated with the risk of more advanced colorectal cancers at diagnosis. OBJECTIVE To evaluate whether the SARS-CoV-2 pandemic was associated with more advanced oncologic stage and change in clinical presentation for patients with colorectal cancer. DESIGN, SETTING, AND PARTICIPANTS This retrospective, multicenter cohort study included all 17 938 adult patients who underwent surgery for colorectal cancer from March 1, 2020, to December 31, 2021 (pandemic period), and from January 1, 2018, to February 29, 2020 (prepandemic period), in 81 participating centers in Italy, including tertiary centers and community hospitals. Follow-up was 30 days from surgery. EXPOSURES Any type of surgical procedure for colorectal cancer, including explorative surgery, palliative procedures, and atypical or segmental resections. MAIN OUTCOMES AND MEASURES The primary outcome was advanced stage of colorectal cancer at diagnosis. Secondary outcomes were distant metastasis, T4 stage, aggressive biology (defined as cancer with at least 1 of the following characteristics: signet ring cells, mucinous tumor, budding, lymphovascular invasion, perineural invasion, and lymphangitis), stenotic lesion, emergency surgery, and palliative surgery. The independent association between the pandemic period and the outcomes was assessed using multivariate random-effects logistic regression, with hospital as the cluster variable. RESULTS A total of 17 938 patients (10 007 men [55.8%]; mean [SD] age, 70.6 [12.2] years) underwent surgery for colorectal cancer: 7796 (43.5%) during the pandemic period and 10 142 (56.5%) during the prepandemic period. Logistic regression indicated that the pandemic period was significantly associated with an increased rate of advanced-stage colorectal cancer (odds ratio [OR], 1.07; 95%CI, 1.01-1.13; P = .03), aggressive biology (OR, 1.32; 95%CI, 1.15-1.53; P < .001), and stenotic lesions (OR, 1.15; 95%CI, 1.01-1.31; P = .03). CONCLUSIONS AND RELEVANCE This cohort study suggests a significant association between the SARS-CoV-2 pandemic and the risk of a more advanced oncologic stage at diagnosis among patients undergoing surgery for colorectal cancer and might indicate a potential reduction of survival for these patients

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Analytical approaches in Alzheimer\u2019s disease Drug Discovery

The research efforts in drug discovery field are based on the knowledge of the molecular aspects of the disease and on the development of new techniques necessary to investigate the biological systems at molecular level. The selection of new leads is therefore a challenging task and involve various essential steps, the first being the identification/validation of new targets, then the selection of molecules able to bind to the target(s), and finally the study of the effects of hitting the target at molecular, cellular and whole animal level. In the case of Alzheimer\u2019s disease (AD), the most common form of dementia in adults, acetylcholinesterase (AChE) has been the first target for the development of new drugs since the discovery of the cholinergic deficit in the central nervous system. However, basic research showed that cognitive impairment could be due not only to a cholinergic deficit but also to a cascade of biochemical events leading to the accumulation in the brain of proteins such as f-amyloid (Ab) and hyper-phosphorylated tau protein. Important targets are amyloid fibrillogenesis, beta-secretase (BACE1), one of the enzymes which cleave APP (amyloid precursor protein) and GSK3b, a tau protein phosphorylating kinase. On the other hand, other non cholinergic role of AChE in the AD has been discovered: some evidences suggest that AChE peripheral binding site may play a key role in the development of senile plaques, accelerating Ab deposition. Once the disease targets have been selected, the determination of the activity of the new compounds must be carried out quickly and in a way that allows the verification of the design hypothesis. Drug activity is in fact mediated by different types of interactions with specific biological targets and the esteem of these interactions may elucidate the mechanism of action. To this aim, in a first instance, high throughput screening methods (HTS) of a large number of compounds for the selection of few lead compounds are required. Secondly, specific methods, which elucidate the selected compound mechanism of action, must be employed, before the ultimate and most advanced tools, transgenic animal models of the disease, can be used to study the effects of single compounds on the disease phenotype. Here we report the development of purposely designed integrated methodologies to define the multifunctional activity profile for new AD drug discovery. With regard to the assessment of the activity of chemical libraries, the affinity chromatography on HPLC immobilized-enzyme column (or immobilized enzyme reactors, IMER) is one of most promising methodologies for HTS applications. Human recombinant AChE and BACE1 monolithic micro-IMERs (immobilized enzyme reactor) have been developed for on-line automated HT HPLC inhibition studies (IC50 and mechanism of inhibition); secondly, fluorometric, circular dichroism, mass spectrometry, AFM methods were optimised for monitoring the inhibition of AChE-induced Ab fibril formation and the inhibition of spontaneous Ab aggregation, elucidating at which intermediate level of the Ab aggregation cascade the inhibitors halt the process (monomer, soluble oligomers, protofibrils, fibrils). Finally, mass spectrometry combined with UHPLC was applied to investigate the mechanism of action of GSK3b inhibitors. By the application of these integrated approaches, new leads as the prototype of new classes of multifunctional compounds for AD treatment were discovered

ELISA and LC-MS/MS methods for determining cyanobacterial toxins in blue-green algae food supplements

The use of natural products as a diet supplement is increasing worldwide but sometimes is not followed by adequate sanitary controls and analyses. Twenty samples of pills and capsules of lyophilised cyanobacteria (blue-green algae), commercialised in Italy as dietary supplements, were found positive at the Vibrio fischeri bioassay. Further analyses with ELISA and LC-MS/MS methods revealed the presence of four microcystin (MC) analogues, MC-LR, -YR, -LA, -RR and two demethylated forms of MC-RR. The highest total microcystin content was 4.5 and 1.4 mu g g(-1) in pills and capsules, respectively. The ELISA measurements, compared to the LC-MS/MS analyses, showed significantly lower concentrations of microcystins in pills, this confirming a possible ELISA underestimate of mixed microcystins, due to different sensitivities for some toxic analogues

DnaK from Bifidobacterium animalis subsp. lactis is a surface-exposed human plasminogen receptor upregulated in response to bile salts.

Bifidobacterium animalis subsp. lactis lives in the gastrointestinal tract of most mammals, including humans. Recently, for the probiotic strain B. animalis subsp. lactis BI07, a dose-dependent plasminogen-binding activity was demonstrated and five putative plasminogen-binding proteins were identified. Here we investigated the role of surface DnaK as a B. animalis subsp. lactis BI07 plasminogen receptor. DnaK was visualized on the bacterial cell surface by transmission electron microscopy. The His-tagged recombinant DnaK protein showed a high affinity for human plasminogen, with an equilibrium dissociation constant in the nanomolar range. The capability to tolerate physiological concentrations of bile salts is a crucial feature for an intestinal symbiont micro-organism. By proteome analysis we demonstrated that the long-term exposure of B. animalis subsp. lactis BI07 to bile salts results in the upregulation of important surface plasminogen receptors such as DnaK and enolase. Moreover, adaptation of B. animalis subsp. lactis BI07 to physiological concentrations of bile salts significantly increased its capacity to interact with the host plasminogen system. By enhancing the bacterial capacity to interact with the host plasminogen, the gut bile environment may facilitate the colonization of the human host by B. animalis subsp. lactis BI07

Outcomes of long‐term anticoagulant treatment for the secondary prophylaxis of splanchnic venous thrombosis

Splanchnic vein thrombosis (SVT) is an uncommon but potentially life‐threatening disease usually related to different underlying clinical conditions. The risk of SVT recurrences is high over time in patients with an underlying permanent prothrombotic condition. Vitamin K antagonists (VKA) represent the mainstay of treatment for SVT. Data about the efficacy and safety of direct oral anticoagulants (DOACs) are reported in the literature for the treatment of acute SVT, but less is known about their application for the secondary prophylaxis of venous thromboembolism (VTE). The aim of this study was to assess the efficacy and safety of long‐term DOACs therapy in patients at high‐risk of thrombosis, compared to VKA

Determination of phytomarkers in pharmaceutical preparations of Hemidesmus indicus roots by micellar electrokinetic chromatography and high-performance liquid chromatography-mass spectrometry

Micellar electrokinetic chromatography was applied to the determination of the major phytomarkers, namely 2-hydroxy-4-methoxybenzaldehyde, 2-hydroxy-4-methoxybenzoic acid, and 3-hydroxy-4-methoxybenzaldehyde, of Hemidesmus indicus root, an Indian medicinal plant. H. indicus bioactive preparations were analyzed by reverse flow micellar electrokinetic chromatography (MEKC) using sodiumtaurodeoxycholate as the surfactant. A pH 2.5 phosphate buffer (50mM) was supplemented with 65mM of sodium taurodeoxycholate to produce the MEKC pseudostationary phase; because of the suppression of the electroosmotic flow, the migration of the partitioned analytes was toward the capillary anodic end. The use of a short fused-silica capillary (8.5 cm effective length; 50 \ub5m i.d.) allowed the separation of phytomarkers, including vanillin and salicylaldehyde (reported as additional metabolites of H. indicus roots), in less than 8 min. The method showed good validation parameters and was applied to the analysis of methanol extracts and a root decoction of H. indicus, a promising botanical drug. The obtained results were compared to those from an independent high-performance liquid chromatography\u2013mass spectrometry method. The 2-Hydroxy-4-methoxybenzaldehyde, 2-hydroxy-4-methoxybenzoic acid, and 3-hydroxy 4-methoxybenzaldehyde were found in all samples confirming their roles as phytomarkers. The absence of vanillin and salicylaldehyde suggested that these latter compounds should not be regarded as characteristic components of the bioactive preparations from the plant roots