Platelets accumulate in lung lesions of tuberculosis patients and inhibit T-cell responses and Mycobacterium tuberculosis replication in macrophages.

Platelets regulate human inflammatory responses that lead to disease. However, the role of platelets in tuberculosis (TB) pathogenesis is still unclear. Here, we show that patients with active TB have a high number of platelets in peripheral blood and a low number of lymphocytes leading to a high platelets to lymphocytes ratio (PL ratio). Moreover, the serum concentration of different mediators promoting platelet differentiation or associated with platelet activation is increased in active TB. Immunohistochemistry analysis shows that platelets localise around the lung granuloma lesions in close contact with T lymphocytes and macrophages. Transcriptomic analysis of caseous tissue of human pulmonary TB granulomas, followed by Gene Ontology analysis, shows that 53 platelet activation-associated genes are highly expressed compared to the normal lung tissue. In vitro activated platelets (or their supernatants) inhibit BCG-induced T- lymphocyte proliferation and IFN-γ production. Likewise, platelets inhibit the growth of intracellular macrophages of Mycobacterium (M.) tuberculosis. Soluble factors released by activated platelets mediate both immunological and M. tuberculosis replication activities. Furthermore, proteomic and neutralisation studies (by mAbs) identify TGF-ß and PF4 as the factors responsible for inhibiting T-cell response and enhancing the mycobactericidal activity of macrophages, respectively. Altogether these results highlight the importance of platelets in TB pathogenesis.

Transglutaminase 2 Regulates Innate Immunity by Modulating the STING/TBK1/IRF3 Axis.

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-ß 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 IFNß 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-ß production associated with the innate immunity response to the cytosolic presence of both self- and pathogen DNA.

Fatal pulmonary arterial thrombosis in a COVID-19 patient, with asymptomatic history, occurred after swab negativization.

BACKGROUND: A considerable number of SARS-CoV-2 infected individuals could be asymptomatic and don't need medical treatment. The clinical spectrum of SARS-CoV-2 infection ranges from asymptomatic cases, medium-intensity forms with mild to moderate symptoms, to severe ones with bilateral pneumonia and respiratory distress. In cases with severe presentation of SARS-CoV-2 infection, the induction of hypercoagulability is one of the pathophysiological mechanism that can contribute to death. CASE PRESENTATION: Here, we reported autoptic evidences of thrombotic pulmonary arterial fatal lesions in an asymptomatic COVID-19 patient, after swab negativization. Whole body complete post-mortem examination was performed, showing the presence of a large thrombus occluding the main pulmonary artery that was the cause of death. Histopathological analysis showed heterogeneous pattern of pathological changes in the lung tissue with numerous vascular thrombi, inflammatory cardiomyopathy and other histopathological modifications in kidneys, spleen and liver. CONCLUSIONS: This study provides evidences that also asymptomatic patients may be at risk to develop thrombotic complications. An appropriate diagnostic screening for thrombotic complications and the early treatment recommendations of antithrombotic drugs could represent an important topic even in asymptomatic individuals.

Postmortem Findings in Italian Patients With COVID-19: A Descriptive Full Autopsy Study of Cases With and Without Comorbidities.

BACKGROUND: Descriptions of the pathological features of coronavirus disease-2019 (COVID-19) caused by the novel zoonotic pathogen severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emanate from tissue biopsies, case reports, and small postmortem studies restricted to the lung and specific organs. Whole-body autopsy studies of COVID-19 patients have been sparse. METHODS: To further define the pathology caused by SARS-CoV-2 across all body organs, we performed autopsies on 22 patients with COVID-19 (18 with comorbidities and 4 without comorbidities) who died at the National Institute for Infectious Diseases Lazzaro Spallanzani-IRCCS Hospital, Rome, Italy. Tissues from the lung, heart, liver, kidney, spleen, and bone marrow (but not the brain) were examined. Only lung tissues were subject to transmission electron microscopy. RESULTS: COVID-19 caused multisystem pathology. Pulmonary and cardiovascular involvement were dominant pathological features. Extrapulmonary manifestations included hepatic, kidney, splenic, and bone marrow involvement, and microvascular injury and thrombosis were also detected. These findings were similar in patients with or without preexisting medical comorbidities. CONCLUSIONS: SARS-CoV-2 infection causes multisystem disease and significant pathology in most organs in patients with and without comorbidities.

TG2 regulates the heat-shock response by the post-translational modification of HSF1.

Heat-shock factor 1 (HSF1) is the master transcription factor that regulates the response to proteotoxic stress by controlling the transcription of many stress-responsive genes including the heat-shock proteins. Here, we show a novel molecular mechanism controlling the activation of HSF1. We demonstrate that transglutaminase type 2 (TG2), dependent on its protein disulphide isomerase activity, triggers the trimerization and activation of HSF1 regulating adaptation to stress and proteostasis impairment. In particular, we find that TG2 loss of function correlates with a defect in the nuclear translocation of HSF1 and in its DNA-binding ability to the HSP70 promoter. We show that the inhibition of TG2 restores the unbalance in HSF1-HSP70 pathway in cystic fibrosis (CF), a human disorder characterized by deregulation of proteostasis. The absence of TG2 leads to an increase of about 40% in CFTR function in a new experimental CF mouse model lacking TG2. Altogether, these results indicate that TG2 plays a key role in the regulation of cellular proteostasis under stressful cellular conditions through the modulation of the heat-shock response.

Transglutaminase type 2-dependent selective recruitment of proteins into exosomes under stressful cellular conditions.

Numerous studies are revealing a role of exosomes in intercellular communication, and growing evidence indicates an important function for these vesicles in the progression and pathogenesis of cancer and neurodegenerative diseases. However, the biogenesis process of exosomes is still unclear. Tissue transglutaminase (TG2) is a multifunctional enzyme with different subcellular localizations. Particularly, under stressful conditions, the enzyme has been also detected in the extracellular matrix, but the mechanism(s) by which TG2 is released outside the cells requires further investigation. Therefore, the goal of the present study was to determine whether exosomes might be a vehicle for TG2 to reach the extracellular space, and whether TG2 could be involved in exosomes biogenesis. To address this issue, we isolated and characterized exosomes derived from cells either expressing or not TG2, under stressful conditions (i.e. proteasome impairment or expressing a mutated form of huntingtin (mHtt) containing 84 polyglutamine repeats). Our results show that TG2 is present in the exosomes only upon proteasome blockade, a condition in which TG2 interacts with TSG101 and ALIX, two key proteins involved in exosome biogenesis. Interestingly, we found that TG2 favours the assembly of a protein complex including mHtt, ALIX, TSG101 and BAG3, a co-chaperone involved in the clearance of mHtt. The formation of this complex is paralleled by the selective recruitment of mHtt and BAG3 in the exosomes derived from TG2 proficient cells only. Overall, our data indicate that TG2 is an important player in the biogenesis of exosomes controlling the selectivity of their cargo under stressful cellular conditions. In addition, these vesicles represent the way by which cells can release TG2 into the extracellular space under proteostasis impairment.

AMBRA1 and SQSTM1 expression pattern in prostate cancer.

Prostate cancer is among the most commonly diagnosed male diseases and a leading cause of cancer mortality in men. There is emerging evidence that autophagy plays an important role in malignant cell survival and offers protection from the anti-cancer drugs in prostate cancer cells. AMBRA1 and the autophagic protein sequestosome-1 (SQSTM1; p62) expression were evaluated by immunohistochemistry and western blot on tissue samples from both benign and malignant prostatic lesions. The data reported in this pilot study demonstrated an increased expression of AMBRA1 and SQSTM1, which were also associated with an accumulation of LC3II in prostate cancer but not in benign lesion. In the present study we found that: (i) at variance with benign lesion, prostate cancer cells underwent SQSTM1 accumulation, i.e., clearly displayed a defective autophagic process but, also, (ii) prostate cancer accumulated AMBRA1 and (iii) this increase positively correlated with the Gleason score. These results underscore a possible implication of autophagy in prostate cancer phenotype and of AMBRA1 as possible cancer progression biomarker in this malignancy.

Characterization of distinct sub-cellular location of transglutaminase type II: changes in intracellular distribution in physiological and pathological states.

Transglutaminase type II (TG2) is a pleiotropic enzyme that exhibits various activities unrelated to its originally identified functions. Apart from post-translational modifications of proteins (peculiar to the transglutaminase family enzymes), TG2 is involved in diverse biological functions, including cell death, signaling, cytoskeleton rearrangements, displaying enzymatic activities, G-protein and non-enzymatic biological functions. It is involved in a variety of human diseases such as celiac disease, diabetes, neurodegenerative diseases, inflammatory disorders and cancer. Regulatory mechanisms might exist through which cells control multifunctional protein expression as a function of their sub-cellular localization. The definition of the tissue and cellular distribution of such proteins is important for the determination of their function(s). We investigate the sub-cellular localization of TG2 by confocal and immunoelectron microscopy techniques in order to gain an understanding of its properties. The culture conditions of human sarcoma cells (2fTGH cells), human embryonic kidney cells (HEK293(TG)) and human neuroblastoma cells (SK-n-BE(2)) are modulated to induce various stimuli. Human tissue samples of myocardium and gut mucosa (diseased and healthy) are also analyzed. Immuno-gold labeling indicates that TG2 is localized in the nucleus, mitochondria and endoplasmic reticulum under physiological conditions but that this is not a stable association, since different locations or different amounts of TG2 can be observed depending on stress stimuli or the state of activity of the cell. We describe a possible unrecognized location of TG2. Our findings thus provide useful insights regarding the functions and regulation of this pleiotropic enzyme.

Evaluation of the Local and Peripheral Immune Responses in Patients with Cystic Echinococcosis.

BACKGROUND: Cystic echinococcosis (CE) cysts may persist for decades because of immune modulation mechanisms. Here, we characterize the cysts and the blood immune responses in patients with CE. METHODS: We enrolled 61 patients with CE and 19 control subjects. We received tissue samples from seven patients with CE and a control subject requiring liver cystectomy. The immunohistochemistry evaluation of the immune cell subtypes and cytokines in the pericysts and surrounding liver and the antigen B (AgB)-specific response analysis of whole blood were performed. RESULTS: In CE, the pericyst and the surrounding liver parenchyma showed aggregates of CD3+ T lymphocytes, mainly CD4+. B lymphocyte aggregates were present in the liver tissue. Monocytes/granulocytes were rarely observed. Th2 cytokine expression was scarce, whereas IFN-γ expression was present in the CE tissues. The control subject did not show an inflammatory infiltrate. The IL-4-specific response to AgB was increased in the patients with CE compared to the control, and this result was confirmed in a larger cohort (p = 0.003), whereas the IFN-γ-response was similar between the two groups (p = 0.5570). CONCLUSION: In patients with CE, CD4+ lymphocytes infiltrate the pericyst and the surrounding liver tissue with a low IL-4/IL-13 expression level and a moderate IFN-γ expression level; moreover, an IL-4 parasite-specific response is detected in the periphery. These results support adventitia involvement in CE immunopathogenesis.

Mpox as AIDS-defining event with a severe and protracted course: clinical, immunological, and virological implications.

A 59-year-old treatment-naive patient with advanced HIV infection presented with a severe and protracted course of mpox (formerly known as monkeypox) that did not respond to the current mpox treatment options. The patient worsened clinically, and developed new mucocutaneous lesions and necrotic evolution of pre-existing ones, along with multiple bilateral lung nodules and the appearance of a tracheal necrotic lesion. Although severe forms of mpox have been observed in people with severe immune system deficiency, including those with advanced HIV presentation, the immunological mechanisms underlying this observation have not yet been fully explained. To our knowledge, this is the first account of a necrotising mpox in a person living with HIV, with viral shedding for more than 11 months and a comprehensive immunological description. Moreover, we documented the virus' persistence by detecting mpox virus DNA from multiple sites and quantified anti-monkeypox virus IgA, IgM, IgG, and neutralising antibodies in serum samples. The severe HIV-driven immune depression and the presence of other co-infections might skew and impair immune responses, thus contributing to the persistence of monkeypox virus infection. Further investigations of immune responses to monkeypox virus infection in people with severe immunosuppression are required to improve management and prevention.

Toxic effects of expanded ataxin-1 involve mechanical instability of the nuclear membrane.

Ataxin 1 (ATXN1) is the protein involved in spinocerebellar ataxia type 1, one of nine dominantly inherited neurodegenerative diseases triggered by polyglutamine expansion. One of the isolated polyglutamine tracts properties is to interact with lipid bilayers. Here we used a multidisciplinary approach to test whether one of the mechanisms responsible for neuronal degeneration involves the destabilization of the nuclear membrane. We thus analyzed the interaction between ATXN1 and lipid membranes, both on cellular models and on artificial lipid bilayers, comparing pathological expanded polyglutamine and histidine interrupted non-harmful polyglutamine tracts of the same length. The toxicity of the different constructs was tested in transiently transfected COS1 cells. Cells expressing pathological ATXN1 presented a significantly higher frequency of anomalous nuclei with respect to those expressing non-harmful ATXN1. Immunofluorescence and electron microscopy showed severe damage in the nuclear membrane of cells expressing the pathological protein. Atomic force microscopy on artificial membranes containing interrupted and non-interrupted partial ATXN1 peptides revealed a different arrangement of the peptides within the lipid bilayer. Force-distance measurements indicated that membrane fragility increases with the lengthening of the uninterrupted glutamine. Transmembrane electrical measurements were performed on artificial bilayers and on the inner nuclear membrane of ATXN1 full length transfected cells. Both artificial lipid bilayers and cellular models demonstrated the dynamic appearance of ionic pathways. Uninterrupted polyglutamines showed not only a larger ionic flow, but also an increase in the single event conductance. Collectively, our results suggest that expanded ATXN1 may induce unregulated ionic pathways in the nuclear membrane, causing severe damage to the cell.

Autopsies Revealed Pathological Features of COVID-19 in Unvaccinated vs. Vaccinated Patients.

Background: In Italy, by the end of 2021, a new pandemic wave led to increased hospitalizations and death, even in some vaccinated people. We aimed to investigate the death of COVID-19-vaccinated patients who acquired infection and developed severe disease, and to assess differences with fatal COVID-19 in unvaccinated subjects by studying the pathological events triggered by SARS-CoV-2. Methods: Detailed autoptic examination was performed on five fully vaccinated compared to five unvaccinated patients. Histopathological analysis focused on the lung and heart, the two major affected organs. Results: COVID-19 caused, or contributed to death, in all the unvaccinated cases. By contrast, in vaccinated group, pre-existing pathologies played a major role, and death was not COVID-19-related in four out of five patients. These patients did not show the histological features of SARS-CoV-2 lung damage. Diffuse inflammatory macrophages infiltration recently emerged as the main feature of COVID-19 cardiac injury. Interestingly, the most striking difference between the two groups was the absence of increased macrophage infiltration in the heart of vaccinated patients. Conclusions: Results of this study confirm the efficacy of anti-SARS-CoV-2 vaccination in protecting organs from injury and support the need to maintain an adequate immune response by booster dose administration.

ZFP750 affects the cutaneous barrier through regulating lipid metabolism.

An essential function of the epidermis is to provide a physical barrier that prevents the loss of water. Essential mediators of this barrier function include ceramides, cholesterol, and very long chain fatty acids, and their alteration causes human pathologies, including psoriasis and atopic dermatitis. A frameshift mutation in the human ZNF750 gene, which encodes a zinc finger transcription factor, has been shown to cause a seborrhea-like dermatitis. Here, we show that genetic deletion of the mouse homolog ZFP750 results in loss of epidermal barrier function, which is associated with a substantial reduction of ceramides, nonpolar lipids. The alteration of epidermal lipid homeostasis is directly linked to the transcriptional activity of ZFP750. ZFP750 directly and/or indirectly regulates the expression of crucial enzymes primarily involved in the biosynthesis of ceramides. Overall, our study identifies the transcription factor ZFP750 as a master regulator epidermal homeostasis through lipid biosynthesis and thus contributing to our understanding of the pathogenesis of several human skin diseases.

Utility of Liver Biopsy in the Diagnosis and Management of Possible Drug-Induced Liver Injury in Patients Receiving Antituberculosis Therapy: A Retrospective Study.

BACKGROUND: Drug-induced liver injury (DILI) secondary to ATT treatment (TB-DILI) is reported in 2-28% of patients. We present here a series of clinical cases of suspected DILI arising during antituberculosis treatment, studied with the aid of liver biopsy. METHODS: this was a retrospective descriptive study including 10 tuberculosis patients who underwent liver biopsy for suspected TB-DILI at the "Lazzaro Spallanzani" Institute from 2017 to 2022. RESULTS: Ten patients who underwent LB were extracted from the database and included in the retrospective study cohort. According to the clinical classification, eight patients had hepatocellular liver injury, one patient had cholestatic injury, and another had mixed-type injury. Histopathological diagnosis revealed liver damage due to DILI in 5/10 (50%) cases. In one case, liver biopsy showed necrotizing granulomatous hepatitis. CONCLUSIONS: Severe and persistent elevation of hepatic transaminases, hepatic cholestasis despite discontinuation of therapy, and other suspected hepatic conditions are indications for liver biopsy, which remains a valuable tool in the evaluation of selected tuberculosis patients with suspected DILI for many reasons. However, the decision to perform a liver biopsy should be based on clinical judgment, considering the benefits and risks of the procedure.

Lymphofollicular lesions associated with monkeypox (Mpox) virus proctitis.

In the recent 2022 monkeypox (Mpox) global outbreak, cases have been mostly documented among men who have sex with men. Proctitis was reported in almost 14% of cases. In this study, four Mpox-confirmed cases requiring hospitalizations for severe proctitis were characterized by clinical, virological, microbiological, endoscopic, and histological aspects. The study showed the presence of lymphofollicular lesions associated with Mpox virus rectal infection for the first time.

Transglutaminase Type 2-MITF axis regulates phenotype switching in skin cutaneous melanoma.

Skin cutaneous melanoma (SKCM) is the deadliest form of skin cancer due to its high heterogeneity that drives tumor aggressiveness. Melanoma plasticity consists of two distinct phenotypic states that co-exist in the tumor niche, the proliferative and the invasive, respectively associated with a high and low expression of MITF, the master regulator of melanocyte lineage. However, despite efforts, melanoma research is still far from exhaustively dissecting this phenomenon. Here, we discovered a key function of Transglutaminase Type-2 (TG2) in regulating melanogenesis by modulating MITF transcription factor expression and its transcriptional activity. Importantly, we demonstrated that TG2 expression affects melanoma invasiveness, highlighting its positive value in SKCM. These results suggest that TG2 may have implications in the regulation of the phenotype switching by promoting melanoma differentiation and impairing its metastatic potential. Our findings offer potential perspectives to unravel melanoma vulnerabilities via tuning intra-tumor heterogeneity.

The purinergic receptor P2X7 and the NLRP3 inflammasome are druggable host factors required for SARS-CoV-2 infection.

Purinergic receptors and NOD-like receptor protein 3 (NLRP3) inflammasome regulate inflammation and viral infection, but their effects on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remain poorly understood. Here, we report that the purinergic receptor P2X7 and NLRP3 inflammasome are cellular host factors required for SARS-CoV-2 infection. Lung autopsies from patients with severe coronavirus disease 2019 (COVID-19) reveal that NLRP3 expression is increased in host cellular targets of SARS-CoV-2 including alveolar macrophages, type II pneumocytes and syncytia arising from the fusion of infected macrophages, thus suggesting a potential role of NLRP3 and associated signaling pathways to both inflammation and viral replication. In vitro studies demonstrate that NLRP3-dependent inflammasome activation is detected upon macrophage abortive infection. More importantly, a weak activation of NLRP3 inflammasome is also detected during the early steps of SARS-CoV-2 infection of epithelial cells and promotes the viral replication in these cells. Interestingly, the purinergic receptor P2X7, which is known to control NLRP3 inflammasome activation, also favors the replication of D614G and alpha SARS-CoV-2 variants. Altogether, our results reveal an unexpected relationship between the purinergic receptor P2X7, the NLRP3 inflammasome and the permissiveness to SARS-CoV-2 infection that offers novel opportunities for COVID-19 treatment.

May macroglossia in COVID-19 be related not only to angioedema?

SARS-CoV-2 infection can lead to a variety of clinical manifestations. The occurrence of tongue swelling has recently reported in severe cases of COVID-19, and angioedema has suggested as the causative mechanism. Several factors, such as genetic predisposing factor and angiotensin-converting enzyme inhibitors (ACEI) therapies, have proposed to induce angioedema, especially as concerns patients requiring ICU treatments. Nevertheless, the question is still debated and other causes not yet recognized should be considered. Here we present a case of macroglossia occurred in a patient deceased for COVID-19 disease, who had no family history of angioedema and did not receive ACEI as antihypertensive drug. Histological and immune-histochemical analysis revealed tongue muscle atrophy with infiltrating macrophages suggesting repair mechanisms, as seen in nerve injury recovery. These new pathological findings may open new fields of study on the pathogenesis of SARS-CoV-2.

Cysteamine exerts in vitro antiviral activity against the SARS-CoV-2 Delta and Omicron variants.

The novel SARS-CoV-2 variants of concern (VOC) represent a considerable global alarm because their mutations are known to affect transmissibility and cause immune escape. While preventing severe disease and deaths, the available vaccines do not avoid infection; therefore, COVID-19 disease management still requires effective therapies. We have recently reported that the aminothiol cysteamine, a drug already applied to humans, exerts direct antiviral activity against SARS-CoV-2 and has in vitro immunomodulatory effect. To evaluate whether this compound exerts antiviral effects also against SARS-CoV-2 variants, we performed different infected cell-based assays using Wild type, Delta, or Omicron VOC. We found that cysteamine significantly reduces the cytopathic effect induced by SARS-CoV-2 Wild type strain and Delta variant in Vero E6 cells. On the other hand, cysteamine had no effects on the survival of cells infected with the Omicron variant, due to the lack of cytotoxicity on Vero E6 cells, at least when infected at MOI = 0.001 for 72 h. Moreover, cysteamine significantly reduced the production of Wild type, Delta, and Omicron variants as measured by the virus released in the culture media (Vero E6 and Calu-3 cells) and by transmission electron microscopy analysis (Vero E6 cells). Notably, cysteamine is more effective in inhibiting the Omicron rather than Delta or Wild type viruses, with an 80% inhibition of Omicron production compared to 40% of Wild type and Delta variant. Overall, our findings demonstrate that cysteamine exerts direct antiviral actions against SARS-CoV-2 Delta and Omicron variants, in addition to the Wild type virus. Our data further demonstrate that cysteamine is a good candidate as repurposing drug for the treatment of SARS-CoV-2 infection for the present and, likely, the future VOC and, therefore, it would be important to investigate its clinical relevance in randomized clinical trials.

Fatal fulminant HSV-2 myocarditis: A complicated presentation.

Herpes simplex virus type 2 (HSV-2) infection is the leading cause of genital lesions. Infrequently HSV-2 primary infection can spread and involve other tissue and organs, however in immunocompetent individuals extra-genital complications are rare findings. In this report we present a fatal case of fulminant myocarditis and terminal liver involvement, caused by HSV-2 infection, with a unique presentation. Diagnosis was made only post-mortem.