Analysis of melanoma tumor antigens and immune subtypes for the development of mRNA vaccine.

Melanoma has a high degree of malignancy and mortality. While there are some hopeful clinical trials for melanoma treatment in progress, they have not yet to yield significant long-term cure rates. Cancer vaccines including mRNA are currently one of the most promising strategy for tumor immunotherapy. The aim of this study was to analyze the potential tumor antigens in melanoma that could be used to develop mRNA vaccines and identify suitable vaccine populations. The gene expression data and complete clinical information of 471 melanoma samples and 1 normal tissue were retrieved from TCGA. Then, 812 samples of normal skin and their corresponding gene expression data were obtained from GTEx. Overexpressed genes, mutated genes and IRDEGs are used to identify potential tumor antigens. The relationship between the expression level of potential antigen and prognosis was analyzed in GEPIA, and then the immune cell infiltration was estimated based on TIMER algorithm. The expression profiles of IRDEGs were used to identify consensus clusters and immune subtypes of melanoma. Finally, mutational status and immune microenvironment characterization in immune subtypes were analyzed. Five tumor antigens (PTPRC, SIGLEC10, CARD11, LILRB1, ADAMDEC1) were identified as potential tumor antigens according to overexpressed genes, mutated genes and immune-related genes. They were all associated with OS, DFS and APCs. We identified two immune subtypes of melanoma, named IS1 and IS2, which exhibit different clinical features and immune landscapes. Based on the different immune landscape, we may conclude that IS1 is immunophenotypically "cold", while IS2 is "hot". The present research implicates that PTPRC, SIGLEC10, CARD11, LILRB1 and ADAMDEC1 may be the antigenic targets for melanoma mRNA vaccines and IS2 patients may be more effective to these vaccines.

Pyroptosis in the lung and spleen of patients died from COVID-19

The purpose of this study was to investigate the expression of pyroptosis-related factors (NLRP3, IL-18, NF-κB, HMGB-1, and GSDMD) in patients who died of COVID-19. The expression levels of NLRP3, IL-18, NF-κB, HMGB-1, and GSDMD in lung and spleen tissues of the COVID-19 group and the control group were detected by tissue immunofluorescence. The control group includes lung tissues and spleen tissues of two patients who died unexpectedly without SARS-CoV-2 infection, and the COVID-19 group includes the lung and spleen tissues of three patients who died of SARS-CoV-2 virus infection. The positive rates of NF-κB, NLRP3, IL-18, and GSDMD in the lung tissues from the control group and COVID-19 group were 9.8% vs 73.4% (p = 0.000), 5.5% vs 63.6% (p = 0.000), 24.4% vs 76.2% (p = 0.000), and 17.5% and 46.8% (p = 0.000) respectively. The positive rates of NF-κB, NLRP3, IL-18, HMGB-1, and GSDMD in the spleen tissues from the control group and COVID-19 group were 20.6% vs 71.2% (p = 0.000), 18.9% vs 72.0% (p = 0.000), 15.2% vs 64.8% (p = 0.000), 27.6% vs 69.2% (p = 0.000), and 23% and 48.8% (p = 0.000), respectively. The positive rates of SARS-CoV-2 spike protein in the CD68 positive cells of the lung and spleen in the control group and COVID-19 group were 2.5% vs 56.8% (p = 0.000);3.0% vs 64.9% (p = 0.000) respectively. The rates of NF-κB positive nuclei in the control group and COVID-19 group were 13.4% vs 51.4% (p = 0.000) in the lung and 38.2% vs 59.3% (p = 0.000) in the spleen. The rates of HMGB-1 positive cytoplasm in the control and the COVID-19 group were 19.7% vs 50.3% (p = 0.000) in the lung and 12.3% vs 45.2% (p = 0.000) in the spleen. The targets of SARS-CoV-2 are the lung and spleen, where increased macrophages could be involved in the up-regulation of pyroptosis-related inflammatory factors such as NF-κB, HMGB-1, NLRP3, IL-18, and GSDMD.

Cell death and pathological findings of the spleen in COVID-19 patients.

The coronavirus disease 2019(COVID-19) is recognized as systemic inflammatory response syndrome. It was demonstrated that a rapid increase of cytokines in the serum of COVID-19 patients is associated with the severity of disease. However, the mechanisms of the cytokine release are not clear. By using immunofluorescence staining we found that the number of CD11b positive immune cells including macrophages in the spleens of died COVID-19 patients, was significantly higher than that of the control patients. The incidence of apoptosis as measured by two apoptotic markers, TUNEL and cleaved caspase-3, in COVID-19 patients' spleen cells is higher than that in control patients. By double immunostaining CD11b or CD68 and SARS-CoV-2 spike protein, it was found that up to 67% of these immune cells were positive for spike protein, suggesting that viral infection might be associated with apoptosis in these cells. Besides, we also stained the autophagy-related molecules (p-Akt、p62 and BCL-2) in spleen tissues, the results showed that the number of positive cells was significantly higher in COVID-19 group. And compared with non-COVID-19 patients, autophagy may be inhibited in COVID-19 patients. Our research suggest that SARS-CoV-2 may result in a higher rate of apoptosis and a lower rate of autophagy of immune cells in the spleen of COVID-19 patients. These discoveries may increase our understanding of the pathogenesis of COVID-19.

The indicative role of markers for liver injury on the severity and prognosis of coronavirus disease 2019 patients.

BACKGROUND: Liver injury in coronavirus disease 2019 (COVID-19) patients was poorly understood. METHODS: The markers of liver injury, severity of disease and prognosis among 495 COVID-19 patients in Zhongnan Hospital of Wuhan University from 1st January 2019 to 11th March 2019 were retrospectively analyzed. RESULTS: The levels of aspartate aminotransferase (AST) (50.1 ± 38.4 vs. 31.4 ± 39.1, P < 0.001), gamma-glutamyl transpeptidase (GGT) (70.3 ± 70.2 vs. 34.1 ± 34.7, P < 0.001) and fibrinogen-to-albumin-ratio (FAR) (13.4 ± 4.0 vs. 10.4 ± 3.4, P < 0.001) were greater than mild COVID-19 patients, whereas the levels of albumin(35.0 ± 6.2 vs. 39.9 ± 3.7, P < 0.001) and albumin/globulin (A/G) ratio (1.21 ± 0.24 vs. 1.50 ± 0.31, P < 0.001) were lower in severe COVID-19 patients. By comparing the changes of liver injury markers 7-10 days after hospitalization, the level of albumin deteriorated from 35.0 ± 6.2 to 30.20 ± 5.5 (P < 0.001), A/G ratio from 1.21 ± 0.24 to 1.06 ± 0.25 (P < 0.001), and FAR from 13.4 ± 4.0 to 15.4 ± 2.9(P < 0.001) in severe COVID-19 patients, while the changes of albumin, A/G ratio and FAR showed opposite patterns in mild COVID-19 patients. FAR > 12 [2.566 (1.410-4.670), P = 0.012) on admission and changes of albumin >5g/l [22.489 (6.422-78.757), P = 0.001] were two risk factors for death, and the sensitivity and specificity for the poor prognosis were 80.8% and 64.0%, 82.6% and 76.3%, respectively. CONCLUSION: The levels of AST, GGT, albumin and FAR are correlated with disease severity after severe acute respiratory syndrome coronavirus-2 infection. FAR > 12 on admission and changes of albumin > 5 g/l were good predictors for the prognosis of COVID-19 patients.

Clinical progression and changes of chest CT findings among asymptomatic and pre-symptomatic patients with SARS-CoV-2 infection in Wuhan, China.

OBJECTIVES: To analyze characteristics of asymptomatic/pres-ymptomatic patients with SARS-CoV-2 infection. METHODS: Chest computed tomography(CT), indicators for organ and coagulation function, inflammation cytokines, of asymptomatic/pre-symptomatic patients with SARS-CoV-2 infection were retrospectively analyzed in Zhongnan Hospital of Wuhan University from 20 December 2019, to 8 March 2020. RESULTS: The proportion of normal chest CT in asymptomatic and pre-symptomatic patients with SARS-CoV-2 infection were 35.4% (17/48) and 3.3%(2/61), respectively (P< 0.001). In 17 asymptomatic patients, their images of chest CT maintained normal during the whole course of diseases, while the normal images of chest CT in 2 pre-symptomatic patients progressed to abnormal later (P< 0.001). All the six asymptomatic patients with SARS-CoV-2 infection maintained unilateral lesion, while the proportion was 29.4%(5/17) in pre-symptomatic patients(P= 0.003). Compared with asymptomatic patients, pre-symptomatic COVID-19 patients had worse levels of Lymphocyte count (P= 0.001), Albumin (P= 0.045), Aspartate aminotransferase (P= 0.044), γ-glutamyl transpeptadase (P= 0.016), Globulin (P= 0.036), Creatinine (P= 0.021), Lactate dehydrogenase (P= 0.008), C-reactive protein (P< 0.001), Serum amyloid A (P< 0.001), and Erythrocyte sedimentation rate (P< 0.001). Except for above indicators, Alkaline phosphatase (P= 0.009), Procalcitonin (P= 0.010), and D-dimer(P< 0.001) increased further during periods of symptoms compared with those levels in pre-symptomatic period. CONCLUSION: In early stage after SARS-CoV-2 infection, images of chest CT and blood tests of asymptomatic patients were different from pre-symptomatic patients.

Clinical characteristics of COVID-19 patients with HIV coinfection in Wuhan, China.

BACKGROUND: Information about the impact of HIV coinfection on clinical characteristics of COVID-19 patients remains limited. METHODS: Maximum body temperatures, fever duration, chest CT and viral shedding, lymphocyte counts, and titer of SARS-CoV-2 antibody were compared between COVID-19 patients with and without HIV infection in Zhongnan Hospital of Wuhan University from January 20th to February 14th, 2020. RESULTS: Compared with 53 COVID-19 patients without HIV infection, the patients with SARS-CoV-2 and HIV coinfection had higher maximum body temperatures (38.7°C vs 37.6°C, P = 0.044), longer duration of fever (8.7 ± 4.5 vs 4.2 ± 2.1 days, P = 0.038), longer time to have improvement of chest CT images (22 vs 15 days from the onset of illness, P = 0.011), lower level of SARS-CoV-2 IgG (5.11 ± 32.33 vs 37.45 ± 15.48 AU/ml, P = 0.042). However, no statistically significant difference of duration of SARS-CoV-2 shedding in the two groups was found (12.3 ± 2.6 vs 13.4 ± 2.4 days, , P = 0.813). CONCLUSION: Lower level of CD4+ T lymphocyte counts caused by HIV infection itself might be one of reasons for relatively weak ability to produce SARS-CoV-2 specific antibodies. The effects of anti-HIV drugs in prevention and treatment of COVID-19 appears to be limited.

The role of methylprednisolone on preventing disease progression for hospitalized patients with severe COVID-19.

BACKGROUND: COVID-19 is a public health emergency that is spreading worldwide and seriously affecting the global economy. Data on the effectiveness and safety of the use of methylprednisolone for patients with severe COVID-19 remain limited. METHODS: In this retrospective study, epidemiological, clinical, laboratory, treatment and outcomes data of hospitalized patients with severe COVID-19 in Zhongnan Hospital of Wuhan University from January 1 to 7 March 2020, were collected. Binary logistic regression model was used to analyse risk factors for disease progression from severe COVID-19 illness to critical illness. The effectiveness and safety of the use of methylprednisolone for patients with severe COVID-19 disease were evaluated. RESULTS: The results of the multivariate analysis from 175 patients with severe COVID-19 indicate that the use of methylprednisolone was a protective factor against disease progression from severe to critical illness(P < .001; OR: 0.054 95% CI: 0.017-0.173). Among patients with severe COVID-19 aged < 65 years, both the proportion of patients who progressed to critical illness (42.2% vs 90.0%, P = .000) and the mortality(6.7% vs 30.0%, P = .002) were lower for patients in methylprednisolone group, compared with those in the non-methylprednisolone group, whereas no statistical differences between the methylprednisolone group and the non-methylprednisolone group were found among patients with COVID-19 older than 65 years. Moreover, both the levels of CD4+ T lymphocyte counts (646 vs 463/µL, P = .007) and IL-6 (241.9 vs 82.8 pg/mL, P = .025) were higher among patients with severe COVID-19 aged < 65 years, compared with those patients ≥ 65 years old. CONCLUSION: Data from the limited sample showed that the early use of low or medium doses of methylprednisolone has a positive effect for patients with severe COVID-19 younger than 65 years old, and excessive immune response and cytokine storm may be some of the reasons for the effectiveness.

CT characteristics and diagnostic value of COVID-19 in pregnancy.

OBJECTIVE: To investigate the computed tomography (CT) characteristics and diagnostic value of novel coronavirus pneumonia (NCP or COVID-19) in pregnancy. METHODS: This study included ten pregnant women infected with COVID-19, treated in the Zhongnan Hospital of Wuhan University from January 20, 2020 to February 6, 2020. Clinical and chest CT data were collected and clinical symptoms, laboratory indicators, and CT images were analyzed to explore CT characteristics and diagnostic value for COVID-19 during pregnancy. RESULTS: Laboratory examination showed that white blood cell count was normal in nine patients, and slightly higher in one patient (10.23 × 109). The lymphocyte ratio decreased in two patients by 12% and 14%, respectively. The levels of C-reactive protein was elevated in seven patients (range, 21.16-60.3 mg/L) and the levels of D-dimer was increased in eight patients (range, 507-2141 ng/mL). Six patients had low levels of total protein (range, 35.3-56.5 mg/L). Two patients showed small patchy ground glass opacity (GGO) involving single lung, while eight patients showed multilobe GGO in both the lungs, with partial consolidation. Peripheral and non-peripheral lesion distributions were seen in ten (100%) and four (40%) patients, respectively. There were four patients who had signs of intra-bronchial air-bronchogram, six patients had small bilateral pleural effusions, while none had lymphadenopathy. Dynamic observations were performed in four patients after COVID-19 treatment. Among these four patients, one patient showed normal on the initial examination, and new lesions were observed after 3 days; 1 patient showed progression after 7 days of treatment, with expansion of the lesion area; and the other 2 patients showed improvement after 14 days of treatment, with reduction in the density and area of lesions and appearance of linear opacity. CONCLUSIONS: The CT characteristics of COVID-19 in pregnancy were mainly observed in early and progressive stages, and multiple new lesions were common. And there were consolidations of varying sizes and degrees within the lesion. Moreover, the original ground glass lesions could be fused or partially absorbed. Six patients had small bilateral pleural effusion. In summary, CT scans can play an important role in early screening, dynamic observation, and efficacy evaluation of suspected or confirmed cases of pregnant women with COVID-19.

Two outbreaks of SARS-CoV-2 in department of surgery in a Wuhan hospital.

The outbreak of SARS-CoV-2 in China, attracted a large number of medical staff to help in the city's hospitals and this put them at a high risk of infection. We describe twenty medical staff who were diagnosed with COVID-19 within one week in the department of surgery in a Wuhan hospital. Epidemiological investigation of these cases identified misdiagnosed patients (source of infection), an inappropriate clinical meeting and working without wearing face masks to be the causes of the outbreaks. This report emphasizes the importance of wearing a facemask and applying other standard infection control precautions to protect medical personnel from infection with the virus.