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Highlights • Takotsubo Syndrome (TTS) is increasingly reported in COVID-19 patients • The pathogenic mechanisms underlying TTS in this particular setting are still unknown • Autopsy findings in fatal cases may contribute to understand the possible role of SARS-CoV-2 in the onset of TTS, COVID-19 can involve several organs and systems, often with indirect and poorly clarified mechanisms. Different presentations of myocardial injury have been reported, with variable degrees of severity, often impacting on the prognosis of COVID-19 patients. The pathogenic mechanisms underlying cardiac damage in SARS-CoV-2 infection are under active investigation. We report the clinical and autopsy findings of a fatal case of Takotsubo Syndrome occurring in an 83-year-old patient with COVID-19 pneumonia. The patient was admitted to Emergency Department with dyspnea, fever and diarrhea. A naso-pharyngeal swab test for SARS-CoV-2 was positive. In the following week his conditions worsened, requiring intubation and deep sedation. While in the ICU, the patient suddenly showed ST segment elevation. Left ventricular angiography showed decreased with hypercontractile ventricular bases and mid-apical ballooning, consistent with diagnosis of Takotsubo syndrome (TTS). Shortly after the patient was pulseless. After extensive resuscitation maneuvers, the patient was declared dead. Autopsy revealed a subepicardial hematoma, in absence of myocardial rupture. On histology, the myocardium showed diffuse edema, multiple foci of contraction band necrosis in both ventricles and occasional coagulative necrosis of single cardiac myocytes. Abundant macrophages CD68+ were detected in the myocardial interstitium. The finding of diffuse contraction band necrosis supports the pathogenic role of increased catecholamine levels; the presence of a significant interstitial inflammatory infiltrate, made up by macrophages, remains of uncertain significance.

To analyze risk factors for type 2 diabetes among women attending menopause clinics in Italy for counselling about the menopause.Women attending a network of first-level outpatient menopause clinics in Italy for general counselling about menopause or treatment of menopausal symptoms.Cross-sectional study with no exclusion criteria. Type 2 diabetes was defined according to National Diabetes Data Groups Indications and the fasting blood glucose at an oral glucose tolerance test within the previous year.Out of the 44 694 considered in this analysis, 808 had a diagnosis of diabetes type 2 (1.8%). In comparison with women aged50 years, the multivariate odds ratios (OR) of type 2 diabetes were 1.31 (95% confidence interval (CI), 0.99-1.74) for women aged 50-52 years, 1.66 (95% CI, 1.27-2.17) at 53-56 years and 2.84 (95% CI, 2.20-3.67) in women agedor = 57 years. Type 2 diabetes was less frequently reported in more educated women (OR high school/university vs. primary school = 0.44 (95% CI, 0.36-0.55)). Being overweight was associated with an increased risk of type 2 diabetes. In comparison with women reporting a low level of physical activity, the multivariate OR of type 2 diabetes was 0.67 (95% CI, 0.54-0.84) for women reporting regular physical activity. In comparison with premenopausal women, the multivariate OR of type 2 diabetes was 1.38 (95% CI, 1.03-1.84) in women with natural menopause. This finding was present also after allowing for the potential confounding effect of age. The multivariate OR of diabetes for users of hormonal replacement therapy was 0.58 (95% CI, 0.46-0.73).This large cross-sectional study suggests that postmenopausal women are at higher risk of type 2 diabetes after allowance for the effect of age. Other main determinants of risk of type 2 diabetes in women around menopause were low socioeconomic status and being overweight. Diabetes was found less frequently in those taking hormone replacement therapy.

The nutritional profile of fresh and dried goji berries cultivated in Italy was investigated. The obtained data confirm goji berries as a source of nutritional and healthy components, such as vitamin E, minerals and fibre. Taking into account the Recommended Daily Allowance (RDA) for minerals and vitamins established by the Commission of the European Communities, Goji berries provide significant amounts of dietary fibre and zeaxanthin and can be declared on the label as a potential source of vitamins E and C. Moreover, dried goji berries can be declared as a source of K, P, Cu, Fe Mn, Zn.

We have recently shown that type 2 transglutaminase (TG2) plays a key role in the host’s inflammatory response during bacterial infections. In this study, we investigated whether the enzyme is involved in the regulation of the STING pathway, which is the main signaling activated in the presence of both self- and pathogen DNA in the cytoplasm, leading to type I IFN (IFN I) production. In this study, we demonstrated that TG2 negatively regulates STING signaling by impairing IRF3 phosphorylation in bone marrow–derived macrophages, isolated from wild-type and TG2 knockout mice. In the absence of TG2, we found an increase in the IFN-β production and in the downstream JAK/STAT pathway activation. Interestingly, proteomic analysis revealed that TG2 interacts with TBK1, affecting its interactome composition. Indeed, TG2 ablation facilitates the TBK1–IRF3 interaction, thus indicating that the enzyme plays a negative regulatory effect on IRF3 recruitment in the STING/TBK1 complex. In keeping with these findings, we observed an increase in the IFNβ production in bronchoalveolar lavage fluids from COVID-19–positive dead patients paralleled by a dramatic decrease of the TG2 expression in the lung pneumocytes. Taken together, these results suggest that TG2 plays a negative regulation on the IFN-β production associated with the innate immunity response to the cytosolic presence of both self- and pathogen DNA.

Macroautophagy selectively degrades dysfunctional mitochondria by a process known as mitophagy. Here we demonstrate the involvement of transglutaminase 2 (TG2) in the turnover and degradation of damaged mitochondria. In TG2-ablated cells we observed the presence of a large number of fragmented mitochondria that display decreased membrane potential, downregulation of IF1 along with increased Drp1 and PINK1 levels, two key proteins regulating the mitochondrial fission. Of note, we demonstrate that in healthy mitochondria, TG2 interacts with the dynamic proteins Drp1 and Fis1; interestingly, their interaction is largely reduced upon induction of the fission process by carbonyl cyanide m-chlorophenyl hydrazine (CCCP). In keeping with these findings, mitochondria lacking TG2 are more susceptible to CCCP treatment. As a consequence of accumulation of damaged mitochondria, cells lacking TG2 increased their aerobic glycolysis and became sensitive to the glycolytic inhibitor 2-deoxy-D-glucose (2-DG). In contrast, TG2-proficient cells are more resistant to 2-DG-induced apoptosis as the caspase 3 is inactivated through the enzyme's crosslinking activity. The data presented in this study show that TG2 plays a key role in cellular dynamics and consequently influences the energetic metabolism.

TG2 ablation affects DC maturation and functions leading to a decreased pro-inflammatory response upon endotoxic shock stimulation. DCs play an essential role in the endotoxic shock, and their profound depletion occurs in septic patients and septic mice. TG2−/− mice are more resistant to the endotoxic shock induced by LPS. Here, we studied the cellular and molecular basis of this effect, analyzing the role of the enzyme in DC maturation and function. We show that TG2 is up-regulated drastically during the final, functional maturation of DCs consequent to LPS treatment. In keeping with this finding, the inhibition of the enzyme cross-linking activity determines the impairment of DC function highlighted by wide phenotypic changes associated with a reduced production of cytokines (IL-10, IL-12) after LPS treatment and a lower ability to induce IFN-γ production by naïve T cells. The in vivo analysis of DCs obtained from TG2−/− mice confirmed that the enzyme ablation leads to an impairment of DC maturation and their reduced responsiveness to LPS treatment. In fact, a marked decrease in DC death, TLR4 down-regulation, and impaired up-regulation of MHCII and CD86 were observed in TG2−/− mice. Taken together, these data suggest that TG2 plays an important role in regulating the response of DCs to LPS and could be a candidate target for treating endotoxin-induced sepsis.

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The basis of the cytopathic effect induced by a laboratory strain and several clinical isolates of human immunodeficiency virus (HIV) in human macrophages cultured in the presence of macrophage colony-stimulating factor was studied. Infected macrophages die of necrosis, the consequence of the production of mature virions in infected cells. Cell death can be prevented by antiviral compounds that interfere with the assembly and budding of virions. Programmed cell death (apoptosis), a potential mechanism of HIV-mediated cell death in CD4 T lymphocytes, does not occur in infected macrophages as shown by electron microscopy, cytofluorometric and gel electrophoretic DNA analysis, and nuclear fluorescent staining by Hoechst and terminal dUTP-nick-end-labeling (TUNEL) assay. The data suggest that macrophage killing by HIV may occur in vivo. Thus, combination therapies that include compounds that inhibit the cytopathic effect of HIV in macrophages should be considered for AIDS patients.

Data on immune responses during human Ebola virus disease (EVD) are scanty, due to limitations imposed by biosafety requirements and logistics. A sustained activation of T-cells was recently described but functional studies during the acute phase of human EVD are still missing. Aim of this work was to evaluate the kinetics and functionality of T-cell subsets, as well as the expression of activation, autophagy, apoptosis and exhaustion markers during the acute phase of EVD until recovery. Two EVD patients admitted to the Italian National Institute for Infectious Diseases, Lazzaro Spallanzani, were sampled sequentially from soon after symptom onset until recovery and analyzed by flow cytometry and ELISpot assay. An early and sustained decrease of CD4 T-cells was seen in both patients, with an inversion of the CD4/CD8 ratio that was reverted during the recovery period. In parallel with the CD4 T-cell depletion, a massive T-cell activation occurred and was associated with autophagic/apoptotic phenotype, enhanced expression of the exhaustion marker PD-1 and impaired IFN-gamma production. The immunological impairment was accompanied by EBV reactivation. The association of an early and sustained dysfunctional T-cell activation in parallel to an overall CD4 T-cell decline may represent a previously unknown critical point of Ebola virus (EBOV)-induced immune subversion. The recent observation of late occurrence of EBOV-associated neurological disease highlights the importance to monitor the immuno-competence recovery at discharge as a tool to evaluate the risk of late sequelae associated with resumption of EBOV replication. Further studies are required to define the molecular mechanisms of EVD-driven activation/exhaustion and depletion of T-cells.

Eukaryotic cells are equipped with an efficient quality control system to selectively eliminate misfolded and damaged proteins, and organelles. Abnormal polypeptides that escape from proteasome-dependent degradation and aggregate in the cytosol can be transported via microtubules to inclusion bodies called ‘aggresomes', where misfolded proteins are confined and degraded by autophagy. Here, we show that Type 2 transglutaminase (TG2) knockout mice display impaired autophagy and accumulate ubiquitinated protein aggregates upon starvation. Furthermore, p62-dependent peroxisome degradation is also impaired in the absence of TG2. We also demonstrate that, under cellular stressful conditions, TG2 physically interacts with p62 and they are localized in cytosolic protein aggregates, which are then recruited into autophagosomes, where TG2 is degraded. Interestingly, the enzyme's crosslinking activity is activated during autophagy and its inhibition leads to the accumulation of ubiquitinated proteins. Taken together, these data indicate that the TG2 transamidating activity has an important role in the assembly of protein aggregates, as well as in the clearance of damaged organelles by macroautophagy.

AIMS/BACKGROUND The mechanism of interaction and the role played by the vesicle lipid composition for the selective association between liposomes and liver cells were studied, at the ultrastructural level, by investigating both in situ and in vitro the interaction between hepatocytes, Kupffer and endothelial liver cells with egg-phosphatidylcholine (eggPC) or eggPC/stearylamine (9:1; mol:mol) reverse-phase evaporation (REV) liposomes. METHODS Liver cells from rats, isolated by enzymatic perfusion and purified by differential centrifugation, were incubated, in a rotating bath at 37 degrees C, with liposomes (2.5 mM final liposomal lipid concentration). Cell aliquots were withdrawn and processed for electron microscope observation at fixed time intervals. Parallel experiments were carried out by in situ liver perfusion with liposome suspensions. RESULTS AND CONCLUSIONS Our first conclusions are: 1) lipidic composition affects the rate of liposomes uptake and internalization by hepatocytes; 2) liposome uptake by hepatocytes or Kupffer cells is likely an endocytic process; 3) endothelial cells internalize lipid vesicles as well; 4) liposome uptake was due to a phagocytic activity for all isolated liver cells, while in the in situ observation endothelial cells seem to use another mechanism (fusion); and 5) the rate of internalization is related to the viability of the treated cells. Experimental data seem to indicate that differential behaviour in the internalization of lipid vesicles exists among parenchymal, Kupffer and endothelial liver cells. These differences suggest that clearance of liposomes by these cells involves two mechanisms (i.e., endocytosis or fusion) with different rates of uptake and internalization that facilitate the design of carriers that can deliver drugs preferentially to a specific liver cell type.

In this work, the proliferative activity of chick embryo hepatocytes and its regulation by factors affecting cell growth, such as epidermal growth factor (EGF) and retinoic acid, were studied. The transport of nutritional molecules, like amino acids, was also investigated; in particular, the uptake mediated by the Na+ -dependent system A, known to be under hormonal and growth factor control. Moreover, some steps in the transduction pathway of mitogenic signal were analyzed, both for EGF and retinoic acid treatment. Results obtained suggest that chick embryo hepatocytes growth in an exclusive serum dependent way shows an early sensibility to EGF stimulation as regards to the proliferative activity and amino acid transport, responds to retinoic acid treatment and lack of contact inhibition. Moreover, as far as the signal transduction is concerned, at early stages, they do not seem to be able to utilize the same signal molecules as observed in adult life.

The influence of cell density on expression of the asialoglycoprotein receptor system in primary cultures of rat hepatocytes was evaluated by measuring the level of the receptor specific mRNA. When the hepatocytes are cultured at high cellular density and are not in a proliferative condition, the transcript molecules of the receptor appear increased about 50% with respect to the low plating density, indicating a modulation of asialoglycoprotein receptor expression at transcriptional level. Such control may be dependent on surface molecules involved in cell specific reassociation, since it is well known that cell contacts play a significant regulatory role in differentiated cells.

Physiological elimination of unwanted cells within the organism occurs via cell death by apoptosis and phagocytosis of these cells represents a key event in the apoptotic process. Macrophages, which are the dedicated phagocytes, and other occasionally phagocytic cells ingest the apoptotic cells while they are still intact, thus preventing the leakage of potentially harmful materials from the dying cells. Although evidence has been presented that the elimination of apoptotic bodies from the tissue operates by means of specific recognition systems, the molecular mechanisms by which an apoptotic cell is recognized are poorly understood. Recent data indicate that phagocyte recognition of apoptotic cells involves at least four classes of receptors on the phagocyte surface. On the other side, dying cells may display different signals to signal their status. Exposure of phosphatidyl serine (PS) on the surface of apoptotic lymphocytes triggers their specific recognition and removal by macrophages. Apoptotic thymocytes are also identified by altered lipid packing on their surface. Different populations of macrophages use either the vitronectin receptor or the PS receptor to recognize and remove apoptotic cells. It has been suggested that the asialoglycoprotein and the galactose-specific receptors of healthy hepatocytes and sinusoidal liver cells are implicated in the engulfment of apoptotic hepatocytes, likely in cooperation with other hepatic carbohydrate-specific receptor systems. The purpose of this review is to examine current knowledge of the mechanisms by which phagocytes recognize and ingest apoptotic cells.

The interaction of bovine pancreatic trypsin inhibitor and bovine tryptase, isolated from liver capsule mast cells, was investigated. They form a complex in vitro with a Ki of 5.6 nM at pH 8.0 and are localized within the mast cell granules, as shown by immunogold staining at the electron microscope level. In addition, double immunogold electron microscopy revealed that the inhibitor and the enzyme are present in the same granules, where they occur in clusters; this may be taken as an indication of their interaction in vivo and suggests a physiological role for bovine pancreatic trypsin inhibitor in the regulation of tryptase proteolytic activity.

The expression of the asialoglycoprotein receptor of hepatocytes and the galactose-specific receptors of non-parenchymal liver cells during the onset of apoptosis in liver of rats treated with lead nitrate was studied. During the involution of lead nitrate-induced hyperplasia in rat liver (occurring at 5 days after the injection) a significant increase of asialoglycoprotein receptor (ASGP-R) expression on hepatocytes coincided with the massive death by apoptosis of the same cells. The increase in the receptor expression was sustained by a large increase in the level of its specific mRNA. As a consequence of lead nitrate injection, we also detected a drastic change of the galactose-specific receptor expression and distribution on the surface of rat liver sinusoidal cells. However, the modulation of the receptor expression on the Kupffer cells did not parallel that observed for the ASGP-R: the peak of surface expression measured on hepatocytes always followed the one observed on Kupffer cells. Our data show a first evidence of a receptor modulation during the process of apoptosis. In fact, the entire carbohydrate recognition system of the liver is modulated during the onset of apoptosis induced by lead nitrate injection, but the pattern of modulation depends on the cellular types. We suggest that a physiological role for the hepatic carbohydrate recognition systems is related to the apoptosis of liver.

We studied the simultaneous binding of galactose and mannose-exposing ligands in sinusoidal rat liver cells during development and aging. The galactose-specific receptors were visualized using 17 nm diameter colloidal gold particles coupled with Lactosylated bovine serum albumine (LacBSA), while mannose-specific receptors were localized by means of 5 nm diameter particles adsorbed with mannan. We observed the presence of four different classes of Kupffer cells in relation to the ligands bound. The percentage of each group of Kupffer cells varied in relation to the age of the subject from which the sample was taken. There were few double-labelled cells in the livers from newborn rats, with numbers increasing with age to adulthood, and decreasing again in the older animals. Cells without labelling were in the majority after birth, but they decreased in number up to adulthood and increased again during subsequent aging. The numbers of single-labelled cells did not change significantly during liver maturation. We hypothesize that the exposition of galactose and mannose-specific receptorial systems is regulated by developmental conditions.

We studied the receptor mediated endocytosis of a modified glycoprotein (N-acetylglucosamine-BSA) and mannan in cultured hepatocytes isolated from 19-days-old embryos. The binding sites for molecules exposing terminal N-acetylglucosamine (GlcNac) and mannose residues were localized and quantified at the ultrastructural level by means of protein-gold complexes. The binding sites were found to be randomly distributed as single gold particles on cultured hepatocyte cell surfaces not restricted to specialized areas of the plasma membrane. The gold ligands were internalized following a receptor mediated pathway, which was studied at different interval times (15, 30 and 60 min.) after incubating the cells with the electron dense markers.

We studied the carbohydrate recognition systems of hepatocytes isolated from 16-day-old embryos, 19-day-old embryos and chicks within 24 h of hatching. We localized and quantified at the ultrastructural level the binding sites for glycoproteins exposing terminal N-acetylglucosamine (GlcNAc), mannose and N-acetylgalactosamine (GalNAc) residues by means of protein-gold complexes. Binding sites specific for GlcNAc and mannose residues are present on hepatocytes from embryos and chicks. On the contrary GalNAc specific binding sites are exclusively observed on cells from 16-day-old embryos. The number and distribution of gold particles on hepatocyte cell surfaces depend on the binding sites and the age considered. We describe a modulation in the number of GlcNAc, and mannose specific receptors present on the cell surface between the embryonal stage and neonatal life.

Bovine Cu, Zn superoxide dismutase (SOD), conjugated to colloidal gold, was intravenously administered to rats and its distribution studied by electron microscopy. Liver was the preferential site of accumulation of gold-labelled SOD. Among liver cells types, Kupffer and endothelial cells showed the presence of the protein earlier than hepatocytes. Uptake by kidney showed slower kinetics than liver. No uptake by heart could be detected. The gold-labelled SOD was localized inside coated pits, coated vesicles and other non-coated endocytic compartments. Absence of binding by BSA-gold complexes and competition between free SOD and the gold-labelled one demonstrated the specificity of the uptake process. Our morphological evidences suggest that in vivo internalization of SOD occurs most likely through receptor-mediated endocytosis.