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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Pleural Mesothelial Cells Modulate the Inflammatory/Profibrotic Response During SARS-CoV-2 Infection.

Although lung fibrosis has a major impact in COVID-19 disease, its pathogenesis is incompletely understood. In particular, no direct evidence of pleura implication in COVID-19-related fibrotic damage has been reported so far. In this study, the expression of epithelial cytokeratins and Wilms tumor 1 (WT1), specific markers of mesothelial cells (MCs), was analyzed in COVID-19 and unrelated pleura autoptic samples. SARS-CoV-2 replication was analyzed by RT-PCR and confocal microscopy in MeT5A, a pleura MC line. SARS-CoV-2 receptors were analyzed by RT-PCR and western blot. Inflammatory cytokines from the supernatants of SARS-CoV-2-infected MeT5A cells were analysed by Luminex and ELLA assays. Immunohistochemistry of COVID-19 pleura patients highlighted disruption of pleura monolayer and fibrosis of the sub-mesothelial stroma, with the presence of MCs with fibroblastoid morphology in the sub-mesothelial stroma, but no evidence of direct infection in vivo. Interestingly, we found evidence of ACE2 expression in MCs from pleura of COVID-19 patients. In vitro analysis shown that MeT5A cells expressed ACE2, TMPRSS2, ADAM17 and NRP1, plasma membrane receptors implicated in SARS-CoV-2 cell entry and infectivity. Moreover, MeT5A cells sustained SARS-CoV-2 replication and productive infection. Infected MeT5A cells produced interferons, inflammatory cytokines and metalloproteases. Overall, our data highlight the potential role of pleura MCs as promoters of the fibrotic reaction and regulators of the immune response upon SARS-CoV-2 infection.

Neuropathology and Inflammatory Cell Characterization in 10 Autoptic COVID-19 Brains.

COVID-19 presents with a wide range of clinical neurological manifestations. It has been recognized that SARS-CoV-2 infection affects both the central and peripheral nervous system, leading to smell and taste disturbances; acute ischemic and hemorrhagic cerebrovascular disease; encephalopathies and seizures; and causes most surviving patients to have long lasting neurological symptoms. Despite this, typical neuropathological features associated with the infection have still not been identified. Studies of post-mortem examinations of the cerebral cortex are obtained with difficulty due to laboratory safety concerns. In addition, they represent cases with different neurological symptoms, age or comorbidities, thus a larger number of brain autoptic data from multiple institutions would be crucial. Histopathological findings described here are aimed to increase the current knowledge on neuropathology of COVID-19 patients. We report post-mortem neuropathological findings of ten COVID-19 patients. A wide range of neuropathological lesions were seen. The cerebral cortex of all patients showed vascular changes, hyperemia of the meninges and perivascular inflammation in the cerebral parenchyma with hypoxic neuronal injury. Perivascular lymphocytic inflammation of predominantly CD8-positive T cells mixed with CD68-positive macrophages, targeting the disrupted vascular wall in the cerebral cortex, cerebellum and pons were seen. Our findings support recent reports highlighting a role of microvascular injury in COVID-19 neurological manifestations.

Dying "from" or "with" COVID-19 during the Pandemic: Medico-Legal Issues According to a Population Perspective.

There is still a lack of knowledge concerning the pathophysiology of death among COVID-19-deceased patients, and the question of whether a patient has died with or due to COVID-19 is still very much debated. In Italy, all deaths of patients who tested positive for SARS-CoV-2 are defined as COVID-19-related, without considering pre-existing diseases that may either contribute to or even cause death. Our study included nine subjects from two different nursing homes (Cases 1-4, Group A; Cases 5-9, Group B). The latter included patients who presumably died from CO poisoning due to a heating system malfunction. All subjects tested positive for COVID-19 both ante- and post-mortem and were examined using post-mortem computed tomography prior to autopsy. COVID-19 was determined to be a contributing cause in the deaths of four out of nine subjects (death due to COVID-19; i.e., pneumonia and sudden cardiac death). In the other five cases, for which CO poisoning was identified as the cause of death, the infection presumably had no role in exitus (death with COVID-19). In our attempt to classify our patients as dying with or due to COVID-19, we found the use of complete assessments (both histological analyses and computed tomography examination) fundamental.

Hepatic Failure in COVID-19: Is Iron Overload the Dangerous Trigger?

Liver injury in COVID-19 patients has progressively emerged, even in those without a history of liver disease, yet the mechanism of liver pathogenicity is still controversial. COVID-19 is frequently associated with increased serum ferritin levels, and hyperferritinemia was shown to correlate with illness severity. The liver is the major site for iron storage, and conditions of iron overload have been established to have a pathogenic role in development of liver diseases. We presented here six patients who developed severe COVID-19, with biochemical evidence of liver failure. Three cases were survived patients, who underwent liver biopsy; the other three were deceased patients, who were autopsied. None of the patients suffered underlying liver pathologies. Histopathological and ultrastructural analyses were performed. The most striking finding we demonstrated in all patients was iron accumulation into hepatocytes, associated with degenerative changes. Abundant ferritin particles were found enclosed in siderosomes, and large aggregates of hemosiderin were found, often in close contact with damaged mitochondria. Iron-caused oxidative stress may be responsible for mitochondria metabolic dysfunction. In agreement with this, association between mitochondria and lipid droplets was also found. Overall, our data suggest that hepatic iron overload could be the pathogenic trigger of liver injury associated to COVID-19.

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.

Evidences for lipid involvement in SARS-CoV-2 cytopathogenesis.

The pathogenesis of SARS-CoV-2 remains to be completely understood, and detailed SARS-CoV-2 cellular cytopathic effects requires definition. We performed a comparative ultrastructural study of SARS-CoV-1 and SARS-CoV-2 infection in Vero E6 cells and in lungs from deceased COVID-19 patients. SARS-CoV-2 induces rapid death associated with profound ultrastructural changes in Vero cells. Type II pneumocytes in lung tissue showed prominent altered features with numerous vacuoles and swollen mitochondria with presence of abundant lipid droplets. The accumulation of lipids was the most striking finding we observed in SARS-CoV-2 infected cells, both in vitro and in the lungs of patients, suggesting that lipids can be involved in SARS-CoV-2 pathogenesis. Considering that in most cases, COVID-19 patients show alteration of blood cholesterol and lipoprotein homeostasis, our findings highlight a peculiar important topic that can suggest new approaches for pharmacological treatment to contrast the pathogenicity of SARS-CoV-2.

Dermatological manifestations during COVID-19 and histological picture: Description of two clinical cases.

It is not yet entirely clear what is the relevance of skin symptoms and what clinical implications are related to their appearance in COVID-19 patients. We describe two cases of COVID-19-associated pneumonia, which presented skin manifestations in advanced stage of illness, when nasopharyngeal swabs became negative for SARS-CoV-2. The first case presented erythematous, maculopapular lesions; the second developed petechial, vesicular and blood-encrusted lesions on the limbs. Histopathology documented perivascular lymphocytic infiltrates, with prevalent CD4+ T-cells in both patients. The research of SARS-CoV-2 in tissues with real time RT-PCR was negative. Basal keratinocytes displayed C4d deposits in one case, who developed laboratory signs indicative of a procoagulative condition at the same time as the skin rash. Skin manifestations during SARS-CoV-2 infection seem to be clinically relevant and further studies are necessary to assess if they are linked to systemic complications, lack of viral clearance or cascades of immune responses induced by the virus, even in patients affected by mild pneumonia.

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.

Fatal Takotsubo syndrome in critical COVID-19 related pneumonia.

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. 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.