PD-L1 Overexpression in the Lungs of Subjects Who Died from COVID-19: Are We on the Way to Understanding the Immune System Exhaustion Induced by SARS-CoV-2?

Knowledge of the pathogenic mechanisms of severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2) is certainly a priority for the scientific community. Two main elements are involved in the biology of the most severe forms of coronavirus disease 2019 (COVID-19): the direct cytopathic effect of the virus against the host tissues, and a dysfunction of the immune system, characterized by the exhaustion of T lymphocytes. The exhaustion of T cells in COVID-19 is poorly understand, but some data could suggest a possible role of PD-1/PD-L1 axis. The aim of this study was to evaluate the possible role of PD-L1 expression in the pulmonary tissue in subjects affected by COVID-19. The presence of SARS-CoV-2 in the pulmonary tissue, and its exact location, was indagated by in situ hybridization; the expression of PD-L1 and CD8 in the same tissue was indagated by immunohistochemistry. Overall, PD-L1 resulted diffusely expressed in 70% of the cases, and an intense expression was observed in 43.5% of cases. Diffuse and intense presence of SARS-CoV-2 by in situ hybridization significantly correlated with an intense PD-L1 expression, and with expression of PD-L1 by pneumocytes. PD-L1 is overexpressed in the pulmonary tissue of subjects died from COVID-19, and mainly in subjects with a high viral load. These data suggest a possible role of PD-L1 in the immune system exhaustion at the basis of the severe forms of the disease.

Variable levels of spike and ORF1ab RNA in post-mortem lung samples of SARS-CoV-2-positive subjects: comparison between ISH and RT-PCR.

Post-mortem examination plays a pivotal role in understanding the pathobiology of the SARS-CoV-2; thus, the optimization of virus detection on the post-mortem formalin-fixed paraffin-embedded (FFPE) tissue is needed. Different techniques are available for the identification of the SARS-CoV-2, including reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), in situ hybridization (ISH), and electron microscopy. The main goal of this study is to compare ISH versus RT-PCR to detect SARS-CoV-2 on post-mortem lung samples of positive deceased subjects. A total of 27 samples were analyzed by RT-PCR targeting different viral RNA sequences of SARS-CoV-2, including envelope (E), nucleocapsid (N), spike (S), and open reading frame (ORF1ab) genes and ISH targeting S and Orf1ab. All 27 cases showed the N gene amplification, 22 out of 27 the E gene amplification, 26 out of 27 the S gene amplification, and only 6 the ORF1ab gene amplification. The S ISH was positive only in 12 out of 26 cases positive by RT-PCR. The S ISH positive cases with strong and diffuse staining showed a correlation with low values of the number of the amplification cycles by S RT-PCR suggesting that ISH is a sensitive assay mainly in cases carrying high levels of S RNA. In conclusion, our findings demonstrated that ISH assay has lower sensitivity to detect SARS-CoV-2 in FFPE compared to RT-PCR; however, it is able to localize the virus in the cellular context since it preserves the morphology.

Crime scene investigation and dynamic reconstruction: importance of synergic collaboration between forensic pathology and digital reconstruction for work-related fatalities.

A crime scene is the result of the dynamic interrelation of various factors. Where a fatality is involved, thorough analysis of the scene by a forensic pathologist produces the first data for verifying consistency between the necropsy results and witness testimonies toward defining the dynamics of injury and death. This step is extremely important in identifying any liability. We present the case of a construction worker who fell through a trapdoor between two floors while holding an iron beam at a building site. This case demonstrates the effectiveness of an integrated approach, using both classic forensic investigation techniques and digital 3-D reconstruction: it allowed verification of the dynamics of the injury and provided important information in assessing the employers liability.

Decreased ghrelin and des-acyl ghrelin plasma levels in patients affected by pharmacoresistant epilepsy and maintained on the ketogenic diet.

BACKGROUND & AIMS: The gastric hormones ghrelin and des-acyl ghrelin have been found to be altered in patients treated with antiepileptic drugs. However, it is unknown if these hormones could be modified by other antiepileptic treatments, such as the ketogenic diet. Especially, a reduction in ghrelin levels could be relevant in view of the growth retardation observed under ketogenic diet treatment. For this reason we aimed to determine the changes in ghrelin and des-acyl ghrelin plasma levels in children affected by refractory epilepsy and treated with the ketogenic diet up to 90 days. METHODS: Both peptides were measured by immunoassays in plasma obtained from 16 children. RESULTS: Ghrelin plasma levels were progressively reduced by the ketogenic diet, reaching a minimum corresponding to 42% of basal levels after 90 days of ketogenic diet (P < 0.05, Duncan's test). Des-acyl ghrelin plasma levels were similarly affected, reaching minimal levels at 30 days (65% of basal levels), and maintaining a significant reduction until 90 days after the onset of ketogenic diet (P < 0.01 for both time intervals). No significant changes in growth were observed during the monitored period of ketogenic diet administration. CONCLUSIONS: Ghrelin and des-acyl ghrelin are downregulated by the ketogenic diet in children affected by refractory epilepsy. Although no significant changes in growth were observed during the short time period of our investigation, the reduction in ghrelin availability may explain the reported growth retardation found in children treated with the ketogenic diet in the long-term.

High plasma levels of ghrelin and des-acyl ghrelin in responders to antiepileptic drugs.

OBJECTIVE: To reconsider ghrelin and des-acyl ghrelin plasma levels in children with epilepsy in order to establish a possible relation with response to antiepileptic drugs (AEDs). METHODS: We designed an observational study in which 114 patients with epilepsy were classified as responders (77) or nonresponders (37) and compared to 59 controls. In these patients, we measured ghrelin and des-acyl ghrelin by immunoassays in blood samples obtained after overnight fast. RESULTS: Ghrelin plasma levels were higher (+94%; p < 0.001, Dunn test) in responders compared to controls. Des-acyl ghrelin plasma levels were also higher in the same group (+55%; p < 0.001). In addition, both hormones were unmodified in nonresponders compared to controls. By comparing responders to nonresponders, ghrelin and des-acyl ghrelin, respectively, were +126% (p < 0.001) and +29% (p < 0.001) in patients with a positive response to AEDs. CONCLUSIONS: These results indicate that ghrelin and des-acyl ghrelin plasma levels are especially high in patients with epilepsy who positively respond to AEDs. In view of the anticonvulsant properties of ghrelin and des-acyl ghrelin, we propose that their higher levels could play a role in modulating the response to AEDs. Moreover, these peptides could be promising markers of response to AEDs.

Electrographic Changes Accompanying Recurrent Seizures under Ketogenic Diet Treatment.

The ketogenic diet (KD) is increasingly used to treat epilepsy refractory to antiepileptic drugs and other neurological disorders. In animal models, the KD was found to increase the threshold to seizures induced by different convulsive stimulations. However, in models in which suprathreshold stimuli were used, a paradoxical seizure worsening was consistently observed in KD-fed animals. To better define this phenomenon, we characterized the electrographic response to seizures induced in mice which were treated with the KD, and then corneally stimulated at 6-Hz in four different sessions. We also evaluated the electroencephalogram (EEG) in three patients in which the KD was associated with a paradoxical worsening of epileptic seizures. Although seizures were initially less severe, a remarkable prolongation of the electrographic response was observed in mice receiving the KD from the second session of 6-Hz corneal stimulation and onwards. The EEG was also markedly altered in the presence of progressive seizure aggravation observed in children treated with the KD, specifically one affected by Lennox-Gastaut syndrome and two by type I lissencephaly,. These results suggest that when seizures are induced or recur because of resistance to therapeutic interventions, the KD may change the EEG by potentiating the electrographic epileptic activity.

Involvement of PPARγ in the Anticonvulsant Activity of EP-80317, a Ghrelin Receptor Antagonist.

Ghrelin, des-acyl ghrelin and other related peptides possess anticonvulsant activities. Although ghrelin and cognate peptides were shown to physiologically regulate only the ghrelin receptor, some of them were pharmacologically proved to activate the peroxisome proliferator-activated receptor gamma (PPARγ) through stimulation of the scavenger receptor CD36 in macrophages. In our study, we challenged the hypothesis that PPARγ could be involved in the anticonvulsant effects of EP-80317, a ghrelin receptor antagonist. For this purpose, we used the PPARγ antagonist GW9662 to evaluate the modulation of EP-80317 anticonvulsant properties in two different models. Firstly, the anticonvulsant effects of EP-80317 were studied in rats treated with pilocarpine to induce status epilepticus (SE). Secondly, the anticonvulsant activity of EP-80317 was ascertained in the repeated 6-Hz corneal stimulation model in mice. Behavioral and video electrocorticographic (ECoG) analyses were performed in both models. We also characterized levels of immunoreactivity for PPARγ in the hippocampus of 6-Hz corneally stimulated mice. EP-80317 predictably antagonized seizures in both models. Pretreatment with GW9662 counteracted almost all EP-80317 effects both in mice and rats. Only the effects of EP-80317 on power spectra of ECoGs recorded during repeated 6-Hz corneal stimulation were practically unaffected by GW9662 administration. Moreover, GW9662 alone produced a decrease in the latency of tonic-clonic seizures and accelerated the onset of SE in rats. Finally, in the hippocampus of mice treated with EP-80317 we found increased levels of PPARγ immunoreactivity. Overall, these results support the hypothesis that PPARγ is able to modulate seizures and mediates the anticonvulsant effects of EP-80317.

Progressive Seizure Aggravation in the Repeated 6-Hz Corneal Stimulation Model Is Accompanied by Marked Increase in Hippocampal p-ERK1/2 Immunoreactivity in Neurons.

The 6-Hz corneal stimulation test is used to screen novel antiepileptic molecules to overcome the problem of drug refractoriness. Although recognized as a standard test, it has been evaluated only recently in the attempt to characterize the putative neuronal networks involved in seizures caused by corneal stimulation. In particular, by recording from the CA1 region we previously established that the hippocampus participates to propagation of seizure activity. However, these findings were not corroborated by using markers of neuronal activation such as FosB/ΔFosB antigens. In view of this discrepancy, we performed new experiments to characterize the changes in levels of phosphorylated extracellular signal-regulated kinases1/2 (p-ERK1/2), which are also used as markers of neuronal activation. To this aim, mice underwent corneal stimulation up to three different times, in three sessions separated by an interval of 3 days. To characterize a group in which seizures could be prevented by pharmacological treatment, we also considered pretreatment with the ghrelin receptor antagonist EP-80317 (330 µg/kg). Control mice were sham-treated. Video electrocorticographic (ECoG) recordings were obtained from mice belonging to each group of treatment. Animals were finally used to characterize the immunoreactivity for FosB/ΔFosB and p-ERK1/2 in the hippocampus. As previously shown, FosB/ΔFosB levels were highly increased throughout the hippocampus by the first induced seizure but, in spite of the progressively increased seizure severity, they were restored to control levels after the third stimulation. At variance, corneal stimulation caused a progressive increase in p-ERK1/2 immunoreactivity all over the hippocampus, especially in CA1, peaking in the third session. Predictably, EP-80317 administration reduced both duration and severity of seizures, prevented the increase in FosB/ΔFosB levels in the first session, and partially counteracted the increase in p-ERK1/2 levels in the third session. The vast majority of p-ERK1/2 immunopositive cells were co-labeled with FosB/ΔFosB antibodies, suggesting the existence of a relationship between the investigated markers in a subpopulation of neurons activated by seizures. These findings suggest that p-ERK1/2 are useful markers to define the aggravation of seizures and the response to anticonvulsant treatments. In particular, p-ERK1/2 expression clearly identified the involvement of hippocampal regions during seizure aggravation in the 6-Hz model.

Microglia are less pro-inflammatory than myeloid infiltrates in the hippocampus of mice exposed to status epilepticus.

Activated microglia, astrogliosis, expression of pro-inflammatory cytokines, blood brain barrier (BBB) leakage and peripheral immune cell infiltration are features of mesial temporal lobe epilepsy. Numerous studies correlated the expression of pro-inflammatory cytokines with the activated morphology of microglia, attributing them a pro-epileptogenic role. However, microglia and myeloid cells such as macrophages have always been difficult to distinguish due to an overlap in expressed cell surface molecules. Thus, the detrimental role in epilepsy that is attributed to microglia might be shared with myeloid infiltrates. Here, we used a FACS-based approach to discriminate between microglia and myeloid infiltrates isolated from the hippocampus 24 h and 96 h after status epilepticus (SE) in pilocarpine-treated CD1 mice. We observed that microglia do not express MHCII whereas myeloid infiltrates express high levels of MHCII and CD40 96 h after SE. This antigen-presenting cell phenotype correlated with the presence of CD4(pos) T cells. Moreover, microglia only expressed TNFα 24 h after SE while myeloid infiltrates expressed high levels of IL-1ß and TNFα. Immunofluorescence showed that astrocytes but not microglia expressed IL-1ß. Myeloid infiltrates also expressed matrix metalloproteinase (MMP)-9 and 12 while microglia only expressed MMP-12, suggesting the involvement of both cell types in the BBB leakage that follows SE. Finally, both cell types expressed the phagocytosis receptor Axl, pointing to phagocytosis of apoptotic cells as one of the main functions of microglia. Our data suggests that, during early epileptogenesis, microglia from the hippocampus remain rather immune supressed whereas myeloid infiltrates display a strong inflammatory profile. GLIA 2016 GLIA 2016;64:1350-1362.

A mild form of rituximab-associated lung injury in two adolescents treated for nephrotic syndrome.

Rituximab is used as a steroid/calcineurin inhibitor-saving agent in patients with nephrotic syndrome. Safety is a crucial issue for justifying widespread use of the drug in this clinical setting. Rituximab-associated lung injury (RALI) is a severe and potentially life-threatening complication in oncohaematological and rheumatological patients, while it has only been anecdotally reported in association with idiopathic nephrotic syndrome (2 cases described, 1 with fatal outcome). We describe a benign form of RALI occurring in two adolescents treated with rituximab (single pulse of 375 mg/m(2)) for nephrotic syndrome. Before treatment, the patients were in good clinical condition while receiving a combination of steroids and calcineurin inhibitors (tacrolimus, case 1 and cyclosporine, case 2). The two patients developed full blown RALI (ie, ground-glass lesions on CT, negative bronchoscopy with bronchoalveolar lavage and deficit in diffusion lung CO transfer), 14 and 40 days after rituximab infusion, respectively. Recovery was rapid and complete after administering steroids in case 1 and with no therapy in case 2. We conclude that RALI may occur in stable non-immunocompromised patients with nephrotic syndrome and its frequency may be higher than expected. Clinical presentation may be mild and resolve after steroids, suggesting hypersensitivity as the main mechanism. Rapid recognition and prompt steroid therapy, if needed, are mandatory for resolution.

Repeated 6-Hz Corneal Stimulation Progressively Increases FosB/ΔFosB Levels in the Lateral Amygdala and Induces Seizure Generalization to the Hippocampus.

Exposure to repetitive seizures is known to promote convulsions which depend on specific patterns of network activity. We aimed at evaluating the changes in seizure phenotype and neuronal network activation caused by a modified 6-Hz corneal stimulation model of psychomotor seizures. Mice received up to 4 sessions of 6-Hz corneal stimulation with fixed current amplitude of 32 mA and inter-stimulation interval of 72 h. Video-electroencephalography showed that evoked seizures were characterized by a motor component and a non-motor component. Seizures always appeared in frontal cortex, but only at the fourth stimulation they involved the hippocampus, suggesting the establishment of an epileptogenic process. Duration of seizure non-motor component progressively decreased after the second session, whereas convulsive seizures remained unchanged. In addition, a more severe seizure phenotype, consisting of tonic-clonic generalized convulsions, was predominant after the second session. Immunohistochemistry and double immunofluorescence experiments revealed a significant increase in neuronal activity occurring in the lateral amygdala after the fourth session, most likely due to activity of principal cells. These findings indicate a predominant role of amygdala in promoting progressively more severe convulsions as well as the late recruitment of the hippocampus in the seizure spread. We propose that the repeated 6-Hz corneal stimulation model may be used to investigate some mechanisms of epileptogenesis and to test putative antiepileptogenic drugs.

Neuroactive peptides as putative mediators of antiepileptic ketogenic diets.

Various ketogenic diet (KD) therapies, including classic KD, medium chain triglyceride administration, low glycemic index treatment, and a modified Atkins diet, have been suggested as useful in patients affected by pharmacoresistant epilepsy. A common goal of these approaches is to achieve an adequate decrease in the plasma glucose level combined with ketogenesis, in order to mimic the metabolic state of fasting. Although several metabolic hypotheses have been advanced to explain the anticonvulsant effect of KDs, including changes in the plasma levels of ketone bodies, polyunsaturated fatty acids, and brain pH, direct modulation of neurotransmitter release, especially purinergic (i.e., adenosine) and γ-aminobutyric acidergic neurotransmission, was also postulated. Neuropeptides and peptide hormones are potent modulators of synaptic activity, and their levels are regulated by metabolic states. This is the case for neuroactive peptides such as neuropeptide Y, galanin, cholecystokinin, and peptide hormones such as leptin, adiponectin, and growth hormone-releasing peptides (GHRPs). In particular, the GHRP ghrelin and its related peptide des-acyl ghrelin are well-known controllers of energy homeostasis, food intake, and lipid metabolism. Notably, ghrelin has also been shown to regulate the neuronal excitability and epileptic activation of neuronal networks. Several lines of evidence suggest that GHRPs are upregulated in response to starvation and, particularly, in patients affected by anorexia and cachexia, all conditions in which also ketone bodies are upregulated. Moreover, starvation and anorexia nervosa are accompanied by changes in other peptide hormones such as adiponectin, which has received less attention. Adipocytokines such as adiponectin have also been involved in modulating epileptic activity. Thus, neuroactive peptides whose plasma levels and activity change in the presence of ketogenesis might be potential candidates for elucidating the neurohormonal mechanisms involved in the beneficial effects of KDs. In this review, we summarize the current evidence for altered regulation of the synthesis of neuropeptides and peripheral hormones in response to KDs, and we try to define a possible role for specific neuroactive peptides in mediating the antiepileptic properties of diet-induced ketogenesis.

Three Rett patients with both MECP2 mutation and 15q11-13 rearrangements.

Autism and Rett syndrome, a severe neurological disorder with autistic behavior, are classified as separate disorders on clinical and etiological ground. Rett syndrome is a monogenic X-linked dominant condition due to de novo mutations in the MECP2 gene, whereas autism is a neurodevelopmental and behavioral disorder with complex genetic basis. Maternally inherited duplications on 15q11-q13 are found in a fraction of autistic children suggesting that an abnormal dosage of gene(s) within this region might cause susceptibility to autism. Now we show that three Rett patients are carriers of both a MECP2 mutation and a 15q11-q13 rearrangement, suggesting that there might be a relationship between autism-related genes and the MECP2 gene.