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.

A Descriptive Study on Causes of Death in Hospitalized Patients in an Acute General Hospital of Southern Italy during the Lockdown due to Covid-19 Outbreak.

(1) Background: All deaths that occurred in a hospital of Southern Italy ("San Giuseppe Moscati" Hospital of Avellino) with medium jurisdiction (up to 425,000 citizens approximately) in the period from 9 March to 4 May 2020 were analyzed. The primary endpoint of the study was to analyze the causes of death in the period study. Secondary endpoints included: (1) the assessment of overall mortality in the emergency period compared with the same period of the past years (2018-2019) in the jurisdiction area; (2) the assessment of the amounts of deaths with positive and negative reverse transcription-polymerase chain reaction (RT-PCR) of nasopharyngeal and oropharyngeal swabs; (3) the frequency of clinical and radiological features consistent with Covid-19 infection in negative RT-PCR cases. (2) Methods: Patients' information and laboratory data were collected through the computerized medical record system (My Hospital, Italy) used for the clinical management of all referring patients. Epidemiological, clinical, and radiological data were reviewed along with the results of nasopharyngeal and oropharyngeal swabs. (3) Results: From 9 March to 4 May 2020, 140 deaths (87 males, 53 females) from all causes occurred in total at "San Giuseppe Moscati" Hospital, of which 32 deaths were Covid-19 related. (4) Conclusions: The excess of mortality could be higher than the one reported in the official epidemiological surveys. False negative cases can have a distorting effect on the assessment of the real mortality rate and the excess mortality. Furthermore, many who died from Covid-19 were likely never tested or they had false negative RT-PCR results. Other victims probably died from causes indirectly related to Covid-19.