Direct exposure to SARS-CoV-2 and cigarette smoke increases infection severity and alters the stem cell-derived airway repair response.

Most demographic studies are now associating current smoking status with increased risk of severe COVID-19 and mortality from the disease but there remain many questions about how direct cigarette smoke exposure affects SARS-CoV-2 airway cell infection. We directly exposed mucociliary air-liquid interface (ALI) cultures derived from primary human nonsmoker airway basal stem cells (ABSCs) to short term cigarette smoke and infected them with live SARS-CoV-2. We found an increase in the number of infected airway cells after cigarette smoke exposure as well as an increased number of apoptotic cells. Cigarette smoke exposure alone caused airway injury that resulted in an increased number of ABSCs, which proliferate to repair the airway. But we found that acute SARS-CoV-2 infection or the combination of exposure to cigarette smoke and SARS-CoV-2 did not induce ABSC proliferation. We set out to examine the underlying mechanism governing the increased susceptibility of cigarette smoke exposed ALI to SARS-CoV-2 infection. Single cell profiling of the cultures showed that infected airway cells displayed a global reduction in gene expression across all airway cell types. Interestingly, interferon response genes were induced in SARS-CoV-2 infected airway epithelial cells in the ALI cultures but smoking exposure together with SARS-CoV-2 infection reduced the interferon response. Treatment of cigarette smoke-exposed ALI cultures with Interferon ß-1 abrogated the viral infection, suggesting that the lack of interferon response in the cigarette smoke-exposed ALI cultures allows for more severe viral infection and cell death. In summary, our data show that acute smoke exposure allows for more severe proximal airway epithelial disease from SARS-CoV-2 by reducing the mucosal innate immune response and ABSC proliferation and has implications for disease spread and severity in people exposed to cigarette smoke.

SARS-CoV-2 infection of primary human lung epithelium for COVID-19 modeling and drug discovery.

Coronavirus disease 2019 (COVID-19) is the latest respiratory pandemic resulting from zoonotic transmission of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). Severe symptoms include viral pneumonia secondary to infection and inflammation of the lower respiratory tract, in some cases causing death. We developed primary human lung epithelial infection models to understand responses of proximal and distal lung epithelium to SARS-CoV-2 infection. Differentiated air-liquid interface cultures of proximal airway epithelium and 3D organoid cultures of alveolar epithelium were readily infected by SARS-CoV-2 leading to an epithelial cell-autonomous proinflammatory response. We validated the efficacy of selected candidate COVID-19 drugs confirming that Remdesivir strongly suppressed viral infection/replication. We provide a relevant platform for studying COVID-19 pathobiology and for rapid drug screening against SARS-CoV-2 and future emergent respiratory pathogens. ONE SENTENCE SUMMARY: A novel infection model of the adult human lung epithelium serves as a platform for COVID-19 studies and drug discovery.

ERCC1 mRNA levels complement thymidylate synthase mRNA levels in predicting response and survival for gastric cancer patients receiving combination cisplatin and fluorouracil chemotherapy.

PURPOSE: We have previously shown that relative thymidylate synthase (TS) mRNA levels in primary gastric adenocarcinomas treated with fluorouracil (5-FU) and cisplatin are inversely associated with response and survival. This is a presumed function of TS as a target for 5-FU activity. We now test the hypotheses that the relative mRNA level of the excision repair cross-complementing (ERCC1) gene is inversely associated with response and survival as an independent function of cisplatin efficacy. PATIENTS AND METHODS: Patients had intact, untreated, primary gastric adenocarcinoma cancer and were evaluated for eligibility on a preoperative cisplatin infusion-5-FU protocol. cDNA, derived from primary gastric tumors before chemotherapy, was used to determine ERCC1 mRNA levels, expressed as the ratio of polymerase chain reaction (PCR) product of the ERCC1 gene and the beta-actin gene. RESULTS: The median ERCC1 mRNA level from 38 primary gastric cancers (33 assessable for response) was 5.8 x 10(-3) (range, 1.8 x 10(-3) to 19.5 x 10(-3)). Of 17 responding patients, 13 (76%) were less than or equal to 5.8 x 10(-3) and four were greater than 5.8 x 10(-3) (P = .003). The median survival for patients with ERCC1 mRNA levels less than or equal to 5.8 x 10(-3) has not been reached, whereas for those greater than 5.8 x 10(-3) it was 5.4 months (P = .034). The median TS mRNA level, 3.7 x 10(-3) (range, 0.9 to 18.9) also segregated responsive versus resistant tumors (P = .024). With both ERCC1 and TS mRNA levels below their medians, 11 of 13 patients (85%) responded; with both ERCC1 and TS mRNA levels above their medians, two of 10 patients (20%) responded (P = .003). CONCLUSION: Considered separately, either ERCC1 or TS mRNA levels in a primary gastric adenocarcinoma has a statistically significant relationship to response. ERCC1 mRNA levels have a statistically significant association with survival; in this cohort TS mRNA levels did not reach statistically significant association with survival as in our previous publication. Whether these molecular parameters are independent of each other as predictors of outcome remains to be determined.

Role of retinal pigment epithelium in the development of experimental autoimmune uveitis.

PURPOSE: To determine the role of retinal pigment epithelium in the induction of S-antigen-induced uveitis by administration of sodium iodate (NaIO3) to selectively damage the retinal pigment epithelium. METHODS: Forty-four Lewis rats were injected with 60 micrograms of S antigen in complete Freund's adjuvant. On postimmunization day 9 the rats were separated into four groups: three groups received NaIO3 at doses of 50, 25, and 10 mg/kg body weight, respectively, and the fourth group (control) received diluent. In addition, separate groups of animals (three in each group) received various doses of NaIO3 or diluent. All of the animals were killed on day 6 after NaIO3 injection, and the eyes were enucleated and submitted for light and electron microscopic examination. In addition, two groups of Lewis rats (6 in each group) were immunized with 0.5 ml of guinea pig spinal cord homogenate in complete Freund's adjuvant to induce experimental allergic encephalomyelitis. On postimmunization day 7, one group received NaIO3 at a dose of 50 mg/kg body weight, whereas the other group received diluent. All animals were killed between days 12 and 14, and spinal cord sections were obtained for microscopic examination. RESULTS: In the control group immunized with S antigen, severe (2+ to 4+) uveoretinitis developed in 70% of the animals. In contrast, only 18% of the animals injected with NaIO3 at a dose of 50 mg/kg body weight exhibited disease, and this was a mild (1+) form. The groups injected with 25 mg/kg (1+ to 2+) and with 10 mg/kg (2+ to 3+) of NaIO3 showed a mild to moderate degree of uveoretinitis in 27% and 50% of the animals, respectively. In the remainder of the animals there was no evidence of uveoretinitis. All of the NaIO3-treated animals showed selective necrosis of the retinal pigment epithelium; this was extensive in the higher dose group and focal in the lower dose groups. In the experimental allergic encephalomyelitis model there was no significant difference in incidence or histologic appearance of demyelinating disease in NaIO3- vs diluent-treated groups. CONCLUSIONS: These results indicate that the retinal pigment epithelium may play a role in the initiation and perpetuation of uveitis after sensitization with S antigen. The effect of NaIO3 appears to be localized to the retinal pigment epithelium; it had no effect on immune reactive cells, as evidenced by the development of experimental allergic encephalomyelitis in animals treated with NaIO3.