Distinct effects of asthma and COPD comorbidity on disease expression and outcome in patients with COVID-19.

BACKGROUND: The impacts of chronic airway diseases on coronavirus disease 2019 (COVID-19) are far from understood. OBJECTIVE: To explore the influence of asthma and chronic obstructive pulmonary disease (COPD) comorbidity on disease expression and outcomes, and the potential underlying mechanisms in COVID-19 patients. METHODS: A total of 961 hospitalized COVID-19 patients with a definite clinical outcome (death or discharge) were retrospectively enrolled. Demographic and clinical information were extracted from the medical records. Lung tissue sections from patients suffering from lung cancer were used for immunohistochemistry study of angiotensin-converting enzyme II (ACE2) expression. BEAS-2B cell line was stimulated with various cytokines. RESULTS: In this cohort, 21 subjects (2.2%) had COPD and 22 (2.3%) had asthma. After adjusting for confounding factors, COPD patients had higher risk of developing severe illness (OR: 23.433; 95% CI 1.525-360.135; P < .01) and acute respiratory distress syndrome (OR: 19.762; 95% CI 1.461-267.369; P = .025) than asthmatics. COPD patients, particularly those with severe COVID-19, had lower counts of CD4+ T and CD8+ T cells and B cells and higher levels of TNF-α, IL-2 receptor, IL-10, IL-8, and IL-6 than asthmatics. COPD patients had increased, whereas asthmatics had decreased ACE2 protein expression in lower airways, compared with that in control subjects without asthma and COPD. IL-4 and IL-13 downregulated, but TNF-α, IL-12, and IL-17A upregulated ACE2 expression in BEAS-2B cells. CONCLUSION: Patients with asthma and COPD likely have different risk of severe COVID-19, which may be associated with different ACE2 expression.

Deficiency of Tfh Cells and Germinal Center in Deceased COVID-19 Patients.

The COVID-19 pandemic caused by SARS-CoV2 is characterized by a remarkable variation in clinical severity ranging from a mild illness to a fatal multi-organ disease. Understanding the dysregulated human immune responses in the fatal subjects is critical for management of COVID-19 patients and the pandemic. In this study, we examined the immune cell compositions in the lung tissues and hilar lymph nodes using immunohistochemistry on 6 deceased COVID-19 patients and 4 focal organizing pneumonia (FOP) patients who underwent lung surgery and served as controls. We found a dominant presence of macrophages and a general deficiency of T cells and B cells in the lung tissues from deceased COVID-19 patients. In contrast to the FOP patients, Tfh cells and germinal center formation were largely absent in the draining hilar lymph nodes in the deceased COVID-19 patients. This was correlated with reduced IgM and IgG levels compared to convalescent COVID-19 patients. In summary, our data highlight a defect of germinal center structure in deceased COVID-19 patients leading to an impaired humoral immunity. Understanding the mechanisms of this deficiency will be one of the key points for the management of this epidemic.

Silencing Filamin A Inhibits the Invasion and Migration of Breast Cancer Cells by Up-regulating 14-3-3σ.

Filamin A and 14-3-3-σ are closely associated with the development of breast cancer. However, the exact relationship between them is still unknown. The present study aimed to examine the interaction of filamin A with 14-3-3-σ in the invasion and migration of breast cancer. RNA interference technology was employed to silence filamin A in MDA-MB-231 cells. Real-time PCR and Western blotting were used to detect the expression of filamin A and 14-3-3-σ at mRNA and protein levels, respectively. Double immunofluorescence was applied to show their colocalization morphologically. Wound healing assay and Trans-well assay were used to testify the migration and invasion of MDA-MB-231 cells in filamin A-silenced cells. The results showed that silencing filamin A significantly increased the mRNA and protein levels of 14-3-3σ. In addition, double immunofluorescence displayed that filamin A and 14-3-3σ were predominantly colocalized in the cytoplasm of MDA-MB-231 cells. Silencing filamin A led to the enhanced fluorescence of 14-3-3σ. Furthermore, cell functional experiments showed that silencing filamin A inhibited the migration and invasion of MDA-MB-231 cells in vitro. In conclusion, silencing filamin A may inhibit the invasion and migration of breast cancer cells by upregulating 14-3-3σ.

Meningeal Ewing Sarcoma/Peripheral PNET: Clinicopathological, Immunohistochemical and FISH study of four cases.

Meningeal Ewing Sarcoma (ES)/peripheral primitive neuroectodermal tumor (pPNET) is a rare diagnostically challenging small round cell tumor in the CNS. This study investigates the clinical pathological features of four cases of this tumor from archives of 6 years in our hospital. Patients were within the median age of 21.5 years and male to female ratio was 1:1. The tumors distributed at the supra-tentorial location, posterior fossa and lumbar vertebral canal, usually presenting as the dura-sited nodule or having close connection with the meninges within the cranium or vertebral canal. Histopathologically, small round undifferentiated tumor cells with hypercellularities, scant cytoplasm and inconspicuous nucleoli were observed, although some components such as atypical larger vesicular nuclei, prominent nucleoli of tumor cells, necrotic foci and mesenchymal collagen proliferation forming the lobular structure, were also appreciated. Immunohistochemally, tumor cells displayed membranous positivity of CD99 (4/4), nuclear positivity of FLI-1 (4/4) and NKX2.2 (4/4), negativity of EMA, GFAP and synaptophysin expression. The histochemical PAS staining showed weak positivity in one case. Fluorescence in situ hybridization (FISH) test using EWSR1 (22q12) dual color break apart rearrangement probe showed positive results in two cases. Results suggest that using a panel of immunohistochemical markers, including NKX2.2, CD99, FLI-1, EMA, GFAP and synaptophysin, combined with the supplementary EWSR1 FISH test, helps to define the diagnosis of meningeal ES/pPNET of CNS.

[Expression of fatty acid synthase and its association with HER2 in invasive ductal carcinoma of breast].

OBJECTIVE: To investigate the expression of fatty acid synthase (FAS) in adenosis, atypical ductal epithelial hyperplasia, ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) of breast, and the correlation of FAS expression with HER2 gene amplification in IDC. METHODS: Immunohistochemical EnVision method staining for FAS was performed in 100 cases of breast lesions and 10 normal breast tissues. HER2 gene amplification was detected with FISH in 60 cases of IDC. RESULTS: The cohort included 10 cases of adenosis, 10 atypical ductal epithelial hyperplasia, 20 DCIS (8 high-grade, 9 intermediated-grade and 3 low-grade), and 60 cases of IDC (5 grade 1, 40 grade 2 and 15 grade 3). FAS expression was negative in all 10 normal breast tissues; in the 10 cases of adenosis, strongly positive FAS expression was detected in one case, positive in 2, weakly positive in 4, and negative in 3; in the 10 cases of atypical ductal epithelial hyperplasia, FAS immunohistochemistry showed that 1 was strongly positive, 4 positive, 4 weakly positive, and 1 negative; in the 20 cases of DCIS, FAS immunostaining showed that 12 were strongly positive, 5 positive, 1 weakly positive, and 2 negative; FAS expression showed a clear increasing trend from normal breast tissue, atypical ductal epithelial hyperplasia to DCIS (χ(2) = 42.02, P < 0.01). Likewise, the increasing trend was also demonstrated from adenosis to DCIS (χ(2) = 34.69, P < 0.01). There was also a positive correlation between FAS expression and extent of lesion among normal breast tissue, adenosis, atypical ductal epithelial hyperplasia and DCIS (χ(2) = 86.02, P < 0.01; r = 0.568, P < 0.01). FAS expression was not correlated with the grade of DCIS (χ(2) = 9.12, P = 0.16). In the five cases of grade 1 IDC, FAS immunostaining showed that 4 cases were strongly positive and 1 positive; in the 40 cases of grade 2 IDC, FAS immunostaining showed that 27 strongly positive, 12 positive, and 1 negative; in the 15 cases of grade 3 IDC, FAS immunostaining showed that 6 were strongly positive, 5 positive, 3 weakly positive, and 1 negative; FAS expression was stronger and more extensive in DCIS, IDC grades 1 and 2 than that in other groups. However, FAS expression was weaker in the IDC grade 3 (χ(2) = 11.26, P = 0.01). The positive expression rate of FAS in IDC was generally higher than that in benign breast lesions (χ(2) = 47.19, P < 0.01). In the 60 cases of IDC, FISH showed HER2 gene amplification in 22 cases, but not in the remaining 38 cases. FAS expression in IDC was highly correlated with HER2 gene amplification (r = 0.44, P < 0.01). The expression of FAS had significant correlation with status of ER and PR and tumor size (P < 0.05). There was no significant correlation with age, immunohistochemical HER2 expression, lymph node metastasis and clinical stage (P > 0.05). CONCLUSIONS: FAS may be closely related to the carcinogenesis of breast IDC. FAS expression is closely associated with HER2 gene amplification in IDC.

Valproic acid attenuates the multiple-organ dysfunction in a rat model of septic shock.

BACKGROUND: Valproic acid (VPA) improves early survival and organ function in a highly lethal poly-trauma and hemorrhagic shock model or other severe insults. We assessed whether VPA could improve organ function in a rat model of septic shock and illustrated the possible mechanisms. METHODS: Forty Sprague-Dawley rats were randomly assigned to four groups (n = 10): control group, VPA group, LPS group, and LPS + VPA group. Lipopolysaccharide (LPS) (10 mg/kg) was injected intravenously to replicate the experimental model of septic shock. Rats were treated with VPA (300 mg/kg, i.v.) or saline. Six hours after LPS injection, blood was sampled for gas analysis, measurement of serum alanine aminotransferase, aspartate aminotransferase, urine nitrogen, creatinine and tumor necrosis factor-alpha. Lung, liver and kidney were collected for histopathological assessment. In addition, myeloperoxidase activity and tumor necrosis factor-a in pulmonary tissue were measured. Acetylation of histone H3 in lung was also evaluated by Western blotting. RESULTS: LPS resulted in a significant decrease in PaO2, which was increased by VPA administration followed LPS injection. In addition, LPS also induced an increase in the serum levels of alanine aminotransferase, aspartate aminotransferase, urine nitrogen, creatinine, and tumor necrosis factor-alpha. However, these increases were attenuated in the LPS + VPA group. The lungs, liver and kidneys from the LPS group were significantly damaged compared with the control group. However, the damage was attenuated in the LPS + VPA group. Myeloperoxidase activity and tumor necrosis factor-alpha levels in pulmonary tissue increased significantly in the LPS group compared with the control group. These increases were significantly inhibited in the LPS + VPA group. Acetylation of histone H3 in lung tissue in the LPS group was inhibited compared with the control. However, the level of acetylation of histone H3 in the LPS + VPA group was markedly elevated in contrast to the LPS group. CONCLUSIONS: Treatment with VPA can attenuate multiple organ damage caused by LPS induced septic shock. Our data also suggest that the beneficial effects are in part due to the decrease in inflammatory cytokines and restoration of normal acetylation homeostasis.

Neuroprotective effect of lipoxin A4 methyl ester in a rat model of permanent focal cerebral ischemia.

Neuroprotective effect of lipoxin A(4) methyl ester (LXA(4) ME) was tested in a rat model of permanent middle cerebral artery occlusion. LXA(4) ME was administrated through intracerebroventricular injection immediately after middle cerebral artery was occluded. Administration of LXA(4) ME ameliorated neurological deficit, reduced infarct volume, attenuated histological damage, and decreased number of apoptotic neuron induced by ischemic insult. These neuroprotective effects of LXA(4) ME were associated with inhibition of neutrophil infiltration, lipid peroxidation, and astrocyte activation. In addition, LXA(4) ME also attenuated proinflammatory cytokines (TNF-alpha and IL-1beta) production. These data suggest that LXA(4) ME protects neuron against permanent cerebral ischemia by inhibiting inflammatory responses.

Melatonin reduces acute lung injury in endotoxemic rats.

BACKGROUND: Treatment with melatonin significantly reduces lung injury induced by bleomycin, paraquat and ischemia reperfusion. In the present study, we investigated the possible protective roles of melatonin in pulmonary inflammation and lung injury during acute endotoxemia. METHODS: Thirty-two male Sprague-Dawley rats were randomly assigned to four groups: vehicle + saline group, melatonin + saline group, vehicle + lipopolysaccharide group, melatonin + lipopolysaccharide group. The rats were treated with melatonin (10 mg/kg, intraperitoneal injection (i.p.)) or vehicle (1% ethanol saline), 30 minutes prior to lipopolysaccharide administration (6 mg/kg, intravenous injection). Four hours after lipopolysaccharide injection, samples of pulmonary tissue were collected. Blood gas analysis was carried out. Optical microscopy was performed to examine pathological changes in lungs and lung injury score was assessed. Wet/dry ratios (W/D), myeloperoxidase activity, malondialdehyde concentrations and tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) levels in lungs were measured. The pulmonary expression of nuclear factor-kappa B (NF-kappaB) p65 was evaluated by Western blotting. RESULTS: PaO(2) in the vehicle + lipopolysaccharide group decreased compared with that in the vehicle + saline group. This decrease was significantly reduced in the melatonin + lipopolysaccharide group. The lung tissues from the saline + lipopolysaccharide group were significantly damaged, which were less pronounced in the melatonin + lipopolysaccharide group. The W/D ratio increased significantly in the vehicle + lipopolysaccharide group (6.1 +/- 0.18) as compared with that in the vehicle + saline group (3.61 +/- 0.3) (P < 0.01), which was significantly reduced in the melatonin + lipopolysaccharide group (4.8 +/- 0.25) (P < 0.01). Myeloperoxidase activity and malondialdehyde levels increased significantly in the vehicle + lipopolysaccharide group compared with that in the vehicle + saline group, which was reduced in the melatonin + lipopolysaccharide group. The TNF-alpha level of pulmonary tissue increased significantly in the vehicle + lipopolysaccharide group ((8.7 +/- 0.91) pg/mg protein) compared with that in the vehicle + saline group ((4.3 +/- 0.62) pg/mg protein, P < 0.01). However, the increase of TNF-alpha level of pulmonary tissue was significantly reduced in the melatonin + lipopolysaccharide group ((5.9 +/- 0.56) pg/mg protein, P < 0.01). Pulmonary IL-10 levels were elevated markedly in the vehicle + lipopolysaccharide group in contrast to that in the vehicle + saline group, whereas the elevation was augmented in the melatonin + lipopolysaccharide group. The nuclear localization of p65 increased markedly in the vehicle + lipopolysaccharide group and this enhancement of nuclear p65 expression was much less in the melatonin + lipopolysaccharide group. CONCLUSION: Melatonin reduces acute lung injury in endotoxemic rats by attenuating pulmonary inflammation and inhibiting NF-kappaB activation.

Reduction of pulmonary inflammatory response by erythropoietin in a rat model of endotoxaemia.

BACKGROUND: Erythropoietin elicits protective effects in lung tissue injury induced by ischaemic reperfusion and hyperoxia. We investigated the protective roles of erythropoietin in pulmonary inflammation and lung injury during acute endotoxaemia. METHODS: A total of 32 male Sprague-Dawley rats were randomly assigned to four groups: saline group, erythropoietin + saline group, saline + lipopolysaccharide group and erythropoietin + lipopolysaccharide group. Rats were treated with erythropoietin (3000 U/kg, i.p.) or saline, 30 minutes prior to lipopolysaccharide administration (6 mg/kg, i.v.). Four hours after lipopolysaccharide injection, samples of pulmonary tissue were collected. Optical microscopy was performed to examine pathological changes in lungs. Wet/dry (W/D) ratios, myeloperoxidase activity, malondialdehyde concentrations and tumour necrosis factor-alpha (TNF-alpha) as well as interleukin 1 beta (IL-1beta) levels in lungs were measured. The pulmonary expression of nuclear factor kappaB (NF-kappaB) p65 was evaluated by Western blotting. Differences between the different groups were analysed by one-way analysis of variance (ANOVA). RESULTS: The lung tissues from the saline + lipopolysaccharide group were significantly damaged, which were less pronounced in the erythropoietin + lipopolysaccharide group. The W/D ratio increased significantly in the saline + lipopolysaccharide group (5.75 +/- 0.22) as compared with the saline group (3.85 +/- 0.20) (P < 0.01), which was significantly reduced in the erythropoietin + lipopolysaccharide group (4.50 +/- 0.35) (P < 0.01). Myeloperoxidase activity and malondialdehyde levels increased significantly in the saline + lipopolysaccharide group compared with the saline group, which was reduced in the erythropoietin + lipopolysaccharide group. The TNF-alpha level of pulmonary tissue increased significantly in the saline + lipopolysaccharide group ((9.80 +/- 0.82) pg/mg protein) compared with the saline group ((4.20 +/- 0.42) pg/mg protein, P < 0.01). However, the increase of TNF-alpha level of pulmonary tissue was significantly reduced in the erythropoietin + lipopolysaccharide group ((6.50 +/- 0.66) pg/mg protein, P < 0.01). Similarly, pulmonary IL-1beta levels were elevated markedly in the saline + lipopolysaccharide group in contrast to the saline group, whereas the elevation was much less in the erythropoietin + lipopolysaccharide group. The nuclear localization of p65 increased markedly in the saline + lipopolysaccharide group and this enhancement of nuclear p65 expression was much less in the erythropoietin + lipopolysaccharide group. CONCLUSION: Erythropoietin attenuates pulmonary inflammation and suppresses TNF-alpha and IL-1beta overproduction during acute endotoxaemia, which is partially mediated by inhibition of NF-kappaB.

[Effects of hypoxia on expression of HIF-1alpha,P53, and cyclin D1 in human lung adenocarcinoma cell line A549].

BACKGROUND & OBJECTIVE: Many studies showed that hypoxia inducible factor-1 alpha (HIF-1alpha)was an essential component for hypoxia- induced cell cycle arrest, but the definite mechanism and the degree of HIF-1alpha affecting cell cycle arrest were unknown yet. This study was to explore the probable mechanism of hypoxia-induced tumor cell cycle arrest. METHODS: Human lung adenocarcinoma cell line A549 were divided into 3 groups: 12-h hypoxia group, 24-h hypoxia group, and control group. The hypoxia groups were exposed to hypoxic conditions (37degrees C, 5% CO2, and 2.0% O2) for 12 h, and 24 h, respectively, while control group was exposed to normal oxygen conditions (37 degrees C, 5% CO2, and 21% O2) for 24 h. Flow cytometry was used to measure the distribution of cell cycles and the expression of cyclin D1. The expression of HIF-1alpha, and p53 was detected using immunohistochemistry. RESULTS: (1) The ratio of G(0)/G(1) in 12-h hypoxia group was (70.20+/-3.33)%, and in 24-h hypoxia group was (82.85+/-1.75)%, significantly higher than that in control group [(50.36+/-4.09) %] (F=202.34, P< 0.01).(2) There was significant difference in cyclin D1 expression among 12-h hypoxia group [(80.22+/-1.55)%], 24-h hypoxia group [(73.65+/-2.10)%], and control group [(90.35+/-2.68)%] (F=100.45, P< 0.01). (3) HIF-1alpha expression in 12-hypoxia group, 24-hypoxia group, and control group was 0.16 +/- 0.02, 0.26 +/- 0.05, and 0.01 +/- 0.00, respectively, with significant difference among the 3 groups (F=105.28, P< 0.01). (4) In hypoxia groups, cyclin D1 expression was negatively correlated with G(0)/G(1) arrest (r=-0.91, P< 0.01), HIF-1alpha expression was positively correlated with p53 expression (r=0.84, P< 0.01), and negatively correlated with cyclin D1 expression (r=-0.90, P< 0.01), and p53 expression was negatively correlated with cyclin D1 expression (r=-0.78,P< 0.01). CONCLUSIONS: Hypoxia can cause G(0)/G(1) cell cycle arrest in human lung adenocarcinoma cell line A549. HIF-1alpha-p53-cyclin D1 pathway might play an important role in the hypoxia-induced G(0)/G(1) cell cycle arrest of human lung adenocarcinoma cell line A549.

[Effects of hypoxia on expression of P-glycoprotein and multidrug resistance protein in human lung adenocarcinoma A549 cell line].

OBJECTIVE: To study the effects of hypoxia on expression of P-gp and multidrug resistance protein in human lung adenocarcinoma A549 cell line, and to explore the probable mechanism of hypoxia in tumor cell of MDR. METHODS: Culturing human lung adenocarcinoma A549 cell in hypoxia (2%O2) for 24 h, the expression of hypoxia inducible factor-1alpha, P-gp and multidrug resistance protein was detected by using immunohistochemistry, and after action of adriamycin or cisplatin in hypoxia (2%O2) for 24, the cell survival rate was detected by MTT. RESULTS: The expression of hypoxia inducible factor-1alpha, P-gp and multidrug resistance protein in hypoxia were higher than the expression in anoxia, and correlation between the expression of HIF-1alpha and P-gp or multidrug resistance protein was observed (P < 0.05). The resistance of adriamycin of A549 cell was enhanced in hypoxia. CONCLUSION: The resistance of tumor chemotherapy is enhanced in hypoxia. The expression of HIF-1alpha is obviously correlated with the expression of P-gp and MRP in A549 cell.