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Liver fibrosis of different etiologies is a serious health problem worldwide. There is no effective therapy available for liver fibrosis except the removal of the underlying cause of injury or liver transplantation. Development of liver fibrosis is caused by fibrogenic myofibroblasts that are not present in the normal liver, but rather activate from liver resident mesenchymal cells in response to chronic toxic or cholestatic injury. Many studies indicate that liver fibrosis is reversible when the causative agent is removed. Regression of liver fibrosis is associated with the disappearance of activated myofibroblasts and resorption of the fibrous scar. In this review, we discuss the results of genetic tracing and cell fate mapping of hepatic stellate cells and portal fibroblasts, their specific characteristics, and potential phenotypes. We summarize research progress in the understanding of the molecular mechanisms underlying the development and reversibility of liver fibrosis, including activation, apoptosis, and inactivation of myofibroblasts.

Here, we present a protocol for isolating human hepatocytes and neural progenitor cells from normal and nonalcoholic steatohepatitis livers. We describe steps for perfusion for scaled-up liver cell isolation and optimization of chemical digestion to achieve maximal yield and cell viability. We then detail a liver cell cryopreservation and potential applications, such as the use of human liver cells as a tool to link experimental and translational research.

Goblet cells (GCs) are specialised guardians lining the intestine. They play a critical role in gut defence and immune regulation. GCs continuously secrete mucus creating a physical barrier to protect from pathogens while harbouring symbiotic gut bacteria adapted to live within the mucus. GCs also form specialised GC-associated passages in a dynamic and regulated manner to deliver luminal antigens to immune cells, promoting gut tolerance and preventing inflammation. The composition of gut bacteria directly influences GC function, highlighting the intricate interplay between these components of a healthy gut. Indeed, imbalances in the gut microbiome can disrupt GC function, contributing to various gastrointestinal diseases like colorectal cancer, inflammatory bowel disease, cystic fibrosis, pathogen infections and liver diseases. This review explores the interplay between GCs and the immune system. We delve into the underlying mechanisms by which GC dysfunction contributes to the development and progression of gastrointestinal diseases. Finally, we examine current and potential treatments that target GCs and represent promising avenues for further investigation.

Liver fibrosis of different etiologies is a serious health problem worldwide. There is no effective therapy available for liver fibrosis except the removal of the underlying cause of injury or liver transplantation. Development of liver fibrosis is caused by fibrogenic myofibroblasts that are not present in the normal liver, but rather activate from liver resident mesenchymal cells in response to chronic toxic or cholestatic injury. Many studies indicate that liver fibrosis is reversible when the causative agent is removed. Regression of liver fibrosis is associated with the disappearance of activated myofibroblasts and resorption of the fibrous scar. In this review, we discuss the results of genetic tracing and cell fate mapping of hepatic stellate cells and portal fibroblasts, their specific characteristics, and potential phenotypes. We summarize research progress in the understanding of the molecular mechanisms underlying the development and reversibility of liver fibrosis, including activation, apoptosis, and inactivation of myofibroblasts.

PurposeTo determine and compare the serum levels of complement Factor H (FH), monomeric C-Reactive Protein (mCRP) and pentameric C-Reactive protein (pCRP) in patients with age-related macular degeneration (AMD) and to correlate them with clinical, structural and functional parameters.MethodsCross-sectional observational study. One hundred thirty-nine individuals (88 patients and 51 healthy controls) from two referral centers were included and classified into three groups: early or intermediate AMD (n=33), advanced AMD (n=55), and age and sex matched healthy controls (n=51). Serum levels of FH, mCRP, and pCRP were determined and correlated with clinical and imaging parameters.ResultsPatients with intermediate AMD presented FH levels significantly lower than controls [186.5 (72.1-931.8) µg/mL vs 415.2 (106.1-1962.2) µg/mL; p=0.039] and FH levels

Summary: Here, we present a protocol for isolating human hepatocytes and neural progenitor cells from normal and nonalcoholic steatohepatitis livers. We describe steps for perfusion for scaled-up liver cell isolation and optimization of chemical digestion to achieve maximal yield and cell viability. We then detail a liver cell cryopreservation and potential applications, such as the use of human liver cells as a tool to link experimental and translational research. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

SARS-CoV-2 pseudovirus infects human hepatocytes leading to metabolic reprogramming towards glycolysis and impaired mitochondrial activity, and metformin can reduce infection under steatotic conditions.

The innate immune response represents a first-line defense against pathogen infection that has been widely conserved throughout evolution. Using the invertebrate Hirudo verbana (Annelida, Hirudinea) as an experimental model, we show here that the RNASET2 ribonuclease is directly involved in the immune response against Gram-positive bacteria. Injection of lipoteichoic acid (LTA), a key component of Gram-positive bacteria cell wall, into the leech body wall induced a massive migration of granulocytes and macrophages expressing TLR2 (the key receptor involved in the response to Gram-positive bacteria) toward the challenged/inoculated area. We hypothesized that the endogenous leech RNASET2 protein (HvRNASET2) might be involved in the antimicrobial response, as already described for other vertebrate ribonucleases, such as RNase3 and RNase7. In support of our hypothesis, HvRNASET2 was mainly localized in the granules of granulocytes, and its release in the extracellular matrix triggered the recruitment of macrophages toward the area stimulated with LTA. The activity of HvRNASET2 was also evaluated on Staphylococcus aureus living cells by means of light, transmission, and scanning electron microscopy analysis. HvRNASET2 injection triggered the formation of S. aureus clumps following a direct interaction with the bacterial cell wall, as demonstrated by immunogold assay. Taken together, our data support the notion that, during the early phase of leech immune response, granulocyte-released HvRNASET2 triggers bacterial clumps formation and, at the same time, actively recruits phagocytic macrophages in order to elicit a rapid and effective eradication of the infecting microorganisms from inoculated area.

Abstract The core characteristics of multi-wall carbon nanotubes (MWCNTs) are impressive and attractive for technology however, since their production and use is steadily increasing, their environmental dispersion could be potentially hazardous to animal and human health. For this reason, the identification of new methods and of reliable models to better understand MWCNT effects is essential. Here we propose the medicinal leech as an alternative model to assess the effects of MWCNTs on immune system. Our previous studies have already demonstrated that in vivo MWCNT treatment induces the activation of leech’s macrophages. Here we will focus on the direct effects of MWCNTs on these cells by isolating and culturing leech's macrophages by means of the consolidated Matrigel technique, followed by MWCNT in vitro treatment. Our results indicate that MWCNT administration causes both the decrease of cell proliferation rate and the increase of the apoptotic rate. Furthermore, since oxidative stress is linked with inflammation, reactive oxygen species has been evaluated confirming that their production rate increases after MWCNT treatment. Our experimental approaches demonstrate the ability of MWCNTs inducing a powerful inflammatory response and confirm that the medicinal leech is a good alternative model to study the possible harmful effects of any nanomaterial.

Elisa Principi,1,* Rossana Girardello,2,* Antonino Bruno,1,* Isabella Manni,3 Elisabetta Gini,2 Arianna Pagani,1 Annalisa Grimaldi,2 Federico Ivaldi,4 Terenzio Congiu,5 Daniela De Stefano,1 Giulia Piaggio,3 Magda de Eguileor,2 Douglas M Noonan,1,2 Adriana Albini1 1Vascular Biology and Angiogenesis, Scientific and Technology Pole, IRCCS MultiMedica, Milano, 2Department of Biotechnology and Life Sciences, University of Insubria, Varese, 3Department of Research, Advanced Diagnosis and Innovation, Regina Elena National Cancer Institute, Rome, 4Department of Neuroscience, Ophthalmology and Genetics, University of Genoa, Genoa, 5Department of Surgical and Morphological Sciences, University of Insubria, Varese, Italy *These authors contributed equally to this work Abstract: The increasing use of carbon nanotubes (CNTs) in several industrial applications raises concerns on their potential toxicity due to factors such as tissue penetrance, small dimensions, and biopersistence. Using an in vivo model for CNT environmental exposure, mimicking CNT exposition at the workplace, we previously found that CNTs rapidly enter and disseminate in the organism, initially accumulating in the lungs and brain and later reaching the liver and kidneys via the bloodstream in CD1 mice. Here, we monitored and traced the accumulation of single-walled CNTs (SWCNTs), administered systemically in mice, in different organs and the subsequent biological responses. Using the novel in vivo model, MITO-Luc bioluminescence reporter mice, we found that SWCNTs induce systemic cell proliferation, indicating a dynamic response of cells of both bone marrow and the immune system. We then examined metabolic (water/food consumption and dejections), functional (serum enzymes), and morphological (organs and tissues) alterations in CD1 mice treated with SWCNTs, using metabolic cages, performing serum analyses, and applying histological, immunohistochemical, and ultrastructural (transmission electron microscopy) methods. We observed a transient accumulation of SWCNTs in the lungs, spleen, and kidneys of CD1 mice exposed to SWCNTs. A dose- and time-dependent accumulation was found in the liver, associated with increases in levels of aspartate aminotransferase, alanine aminotransferase and bilirubinemia, which are metabolic markers associated with liver damage. Our data suggest that hepatic accumulation of SWCNTs associated with liver damage results in an M1 macrophage-driven inflammation. Keywords: single-walled carbon nanotubes, nanotoxicity, metabolism, hepatic function, inflammation, Kupffer cells, mouse models

Human breast adenocarcinoma cells (MCF7) grow in three-dimensional culture as spheroids that represent the structural complexity of avascular tumors. Therefore, spheroids offer a powerful tool for studying cancer development, aggressiveness, and drug resistance. Notwithstanding the large amount of data regarding the formation of MCF7 spheroids, a detailed description of the morpho-functional changes during their aggregation and maturation is still lacking. In this study, in addition to the already established role of gap junctions, we show evidence of tunneling nanotube (TNT) formation, amyloid fibril production, and opening of large stable cellular bridges, thus reporting the sequential events leading to MCF7 spheroid formation. The variation in cell phenotypes, sustained by dynamic expression of multiple proteins, leads to complex networking among cells similar to the sequence of morphogenetic steps occurring in embryogenesis/organogenesis. On the basis of the observation that early events in spheroid formation are strictly linked to the redox homeostasis, which in turn regulate amyloidogenesis, we show that the administration of N-acetyl-l-cysteine (NAC), a reactive oxygen species (ROS) scavenger that reduces the capability of cells to produce amyloid fibrils, significantly affects their ability to aggregate. Moreover, cells aggregation events, which exploit the intrinsic adhesiveness of amyloid fibrils, significantly decrease following the administration during the early aggregation phase of neutral endopeptidase (NEP), an amyloid degrading enzyme.

Adriana Albini,1,* Arianna Pagani,2,3,* Laura Pulze,2 Antonino Bruno,3 Elisa Principi,3 Terenzio Congiu,4 Elisabetta Gini,2,4 Annalisa Grimaldi,2 Barbara Bassani,2,3 Silvio De Flora,5 Magda de Eguileor,2 Douglas M Noonan2,3 1Laboratory of Translational Research, IRCCS Arcispedale Santa Maria Nuova, Reggio Emilia, 2Department of Biotechnologies and Life Sciences, University of Insubria, Varese, 3Scientific and Technology Park, IRCCS MultiMedica, Milano, 4Department of Surgical and Morphological Sciences, University of Insubria, Varese, 5Department of Health Sciences, University of Genoa, Genoa, Italy *These authors contributed equally to this work Abstract: Carbon nanotubes (CNTs) have been extensively investigated and employed for industrial use because of their peculiar physical properties, which make them ideal for many industrial applications. However, rapid growth of CNT employment raises concerns about the potential risks and toxicities for public health, environment, and workers associated with the manufacture and use of these new materials. Here we investigate the main routes of entry following environmental exposure to multi-wall CNTs (MWCNTs; currently the most widely used in industry). We developed a novel murine model that could represent a surrogate of a workplace exposure to MWCNTs. We traced the localization of MWCNTs and their possible role in inducing an innate immune response, inflammation, macrophage recruitment, and inflammatory conditions. Following environmental exposure of CD1 mice, we observed that MWCNTs rapidly enter and disseminate in the organism, initially accumulating in lungs and brain and later reaching the liver and kidney via the bloodstream. Since recent experimental studies show that CNTs are associated with the aggregation process of proteins associated with neurodegenerative diseases, we investigated whether MWCNTs are able to induce amyloid fibril production and accumulation. Amyloid deposits in spatial association with macrophages and MWCNT aggregates were found in the brain, liver, lungs, and kidneys of exposed animals. Our data suggest that accumulation of MWCNTs in different organs is associated with inflammation and amyloid accumulation. In the brain, where we observed rapid accumulation and amyloid fibril deposition, exposure to MWCNTs might enhance progression of neurodegenerative and other amyloid-related diseases. Our data highlight the conclusion that, in a novel rodent model of exposure, MWCNTs may induce macrophage recruitment, activation, and amyloid deposition, causing potential damage to several organs. Keywords: nanoparticles, animal model, environmental exposure, inflammation, amyloid fibrils, macrophages

Allograft inflammatory factor-1 (AIF-1) is a 17 kDa cytokine-inducible calcium-binding protein that in Vertebrates plays an important role in allografts immune response. Since its expression is mainly limited to the monocyte/macrophage lineage, it was recently suggested that it could play a key role during inflammatory responses, allograft rejection, as well as in the activation of macrophages. To clarify this point we have focused our research on the possible role of AIF-1 during the inflammatory response after injury in the leech Hirudo medicinalis (Annelida, Hirudinea). This invertebrate is an excellent animal model since the responses evoked during inflammation and tissue repair are clear and easily detectable and have a striking similarity with vertebrate responses. Moreover the analysis of an EST library from H. medicinalis CNS, revealed the presence of a gene, named Hmaif-1/alias Hmiba1, showing a high homology with vertebrate aif-1. Our data show that the related protein, named HmAIF-1, is constitutively expressed in unlesioned leeches and that dramatically increases 48 h after wounds and tissue transplants. Immunohistochemistry experiments, using a specific anti HmAIF-1 polyclonal antibody, shows that this factor is present in spread, CD68+ /CD45+ macrophage-like cells. A few days after experimental wounding of the body wall, the amount of these immunopositive cells increases at the lesion site. In conclusion here we propose that in leech HmAIF-1 factor is involved in inflammation events like its vertebrate counterparts.

Owing to its memory and plasticity, the immune system (IS) is capable of recording all the immunological experiences and stimuli it was exposed to. The combination of type, dose, intensity, and temporal sequence of antigenic stimuli that each individual is exposed to has been named “immunobiography.” This immunological history induces a lifelong continuous adaptation of the IS, which is responsible for the capability to mount strong, weak or no response to specific antigens, thus determining the large heterogeneity of immunological responses. In the last years, it is becoming clear that memory is not solely a feature of adaptive immunity, as it has been observed that also innate immune cells are provided with a sort of memory, dubbed “trained immunity.” In this review, we discuss the main characteristics of trained immunity as a possible contributor to inflammaging within the perspective of immunobiography, with particular attention to the phenotypic changes of the cell populations known to be involved in trained immunity. In conclusion, immunobiography emerges as a pervasive and comprehensive concept that could help in understanding and interpret the individual heterogeneity of immune responses (to infections and vaccinations) that becomes particularly evident at old age and could affect immunosenescence and inflammaging.

One of the major alterations observed in mealybug Planococcus citri parasitized by Leptomastix dactylopii is a strong reduction of laid eggs, which is evident soon after parasitization. Venom injection in unparasitized hosts determines a drastic reduction of fecundity indicating that this female secretion injected at the oviposition plays a key-role in host regulation. In order to assess the impact of parasitism and venom injection on host reproductive tissues, ovaries were dissected at different time intervals after these treatments and observed by light and transmission electron microscopy. The developing eggs showed clear symptoms of degeneration, already half an hour after parasitization or venom injection. Heat and protease treatments of venom nearly suppressed its effects on host reproduction, indicating that proteins are likely responsible for the observed alterations. The electrophoretic profile of venom proteins covers a wide range of molecular masses between 15 to 200 kDa but five major bands having a molecular mass of about 27, 30, 40, 90 and 120 kDa respectively were more evident. Moreover, to establish any parasitoid preference in host selection, among the adult female mealybugs at different stages of maturation and a possible relation with fecundity reduction in the host, the parasitoid behavior was observed.

LPS stimulation provokes serious cellular stress with an increase of cytoplasmic reactive oxygen species (ROS). We have investigated, among the different cellular defenses, amyloidogenesis as common physiological response to attenuate oxidative stress. Optical and electron microscopic observations of the following LPS activated cell lines [insect (larval hemocytes, IPLB-LdFB and Drosophila Schneider’s S2 cells); mouse (NIH3T3 embryonic fibroblasts); Human (Human Umbilical Vein Endothelial Cells (HUVEC), neutrophils, and mesenchymal stem cells] reveal that, all are characterized by irregular profiles, cytoplasmic empty vacuoles or by cisternae containing fibrillar material. The compartmentalized fibrillar material shows staining properties typical of amyloid fibrils. LPS activation leads to ROS generation, resulting in pH acidification. Stimulated cells show pink cytoplasm in May-Grünwald Giemsa differential staining, giving a gross indication of a lower intracellular pH. Moreover the activation of amyloidogenesis is also linked with an extensive production of ACTH and α-MSH in all cultured cell types. We suggest that amyloidogenesis is a common, physiological cellular response to weak ROS, starting when other anti-stress cellular systems failed to restore homeostasis. The morphological evidence and/or functional characterization of synthesized amyloid fibrils could be an early indicator of oxidative stress that may lead to a general inflammatory process.

In some leeches like Hirudo medicinalis, any kind of stimulation (surgical wound or growth factor injection) provokes the botryoidal tissue response. This peculiar tissue, localized in the loose connective tissue between gut and body wall, is formed by granular botryoidal cells and flattened endothelial-like cells. Under stimulation, the botryoidal tissue changes its shape to form new capillaries. In mammals, the molecular regulation of the angiogenic phenotype requires coordinated input from a number of signalling molecules: among them the GTPase Ras is one of the major actor. In our current study, we determine whether Ras activation alone would be sufficient to drive vessels formation from leech botryoidal tissue. Our findings indicate that assembly and disassembly of actin filaments regulated by Ras protein is involved in morphological modification of botryoidal tissue cells during leech angiogenic process.