Background: Respiratory viral infections (respiratory syncytial and influenza A viruses) have been found to be associated with deterioration of pulmonary function and exacerbations in people with cystic fibrosis (CF) [1].Conversely, PwCF infected by SARS-CoV-2 are not experiencing worse clinical outcomes than the general population, except for lung-transplanted patients, who are more likely to require oxygen therapy and hospitalization and are six times as likely to die.Several studies have reported that PwCF with SARS-CoV-2 have a lower case fatality rate than the general population [2][3][4].We hypothesized that loss of CF transmembrane conductance regulator (CFTR) protein expression and function may provide an advantage against severe COVID-19 outcomes.Methods: We investigated expression of Angiotensin-converting enzyme 2 (ACE2) and CD13 (ANpEp) messenger ribonucleic acid (mRNA) and protein in CF and non-CF bronchial epithelial (CFBE) cells, including polarized CFBE41o -, 16HBE14o -, Calu-3, and air-liquid interface (ALI)-differentiated primary nasal and bronchial epithelia.Cell susceptibility to SARS-CoV-2 infection was assayed by infecting single cells with a multiplicity of infection of 0.1 for 2 hours.Twenty-four, 48, and 72 hours after infection, RNA was isolated from cells and supernatants, and SARS-CoV-2 titration was obtained using a TaqMan assay by quantitative polymerase chain reaction and digital polymerase chain reaction.IL-6 levels were quantified by enzyme-linked immunosorbent assay in supernatants from primary airway epithelial cells stimulated with 1 mg/mL of SARS-CoV-2 spike protein for 12 hours.Results: Expression of SARS-CoV-2 receptor ACE2 is significantly downregulated in CF cells in terms of mRNA and protein.We consistently observed that expression of the aminopeptidase CD13, which has been found co-expressed with ACE2 in several tissues and acts as a co-receptor for other human coronaviruses, is equally downregulated.We reported that the F508del-mutated CFTR channel promotes mislocalization of ACE2, which is almost completely retained into the endoplasmic reticulum, similar to unfolded CFTR, suggesting an association between these two proteins.Most importantly, lower ACE2 expression in CF cells is associated with less SARS-CoV-2 viral entry and replication.Eventually, spike-induced IL-6 levels were significantly lower in ALI-differentiated primary airway epithelia obtained from people with CF, consistent with the lower ACE2 expression.Conclusions: This study clarifies why SARS-CoV-2 infection does not promote particularly severe outcomes in people with CF, unless they have received lung transplantation.Our results also showcase CFTR as a regulator of ACE2 and SARS-CoV-2 viral entry and replication.

Objectives: Many molecular mediators have been described to possess cancer stem cell (CSC) characteristics in ovarian cancer. It has been hypothesized that these mediators are potentiated by chemotherapy, which portends earlier recurrence and shorter survival. We aimed to explore the effect of chemotherapy on ovarian CSC-like mediators. Methods: Matched pre- and post-chemotherapy tumor specimens from ovarian cancer patients were obtained. All patients underwent neoadjuvant chemotherapy with interval debulking surgery. Samples were analyzed for expression of 27 CSC markers via quantitative polymerase chain reaction (qPCR). Data were depicted as a fold change in gene expression between post- and pre-treatment samples and compared with clinical factors. Associated immunohistochemical stains were used to validate qPCR data. CSC markers were validated in a tumorsphere model and in vivo tumor-initiating studies. Results: Specimens from 22 patients with stage IIIC/IV serous ovarian cancer were obtained. Twenty-seven CSC markers demonstrated an increase in gene expression after exposure to chemotherapy. A 3-fold or greater increase in gene expression after exposure to chemotherapy was seen in 9 (33%) of 27 markers: ABCG2 (5.8-fold), ALDH1A1 (4.0), CTGF (5.4), DPP4 (4.2), MYC (3.4), POSTN (6.7), CD133 (6.5), SOX2 (8.5), and VCAN (3.2). Only 3 markers demonstrated a significant fold increase that correlated with platinum resistance: POSTN 4.1-fold (P = .04), ALDH1A1 5-fold (P = .037), and SOX2 14.5-fold (P = .004). SOX2(hi) OVCAR8 ovarian cancer cells exhibited significantly higher levels of tumorsphere-forming potential (P = .04) than Sox2(lo) cells. SOX2(hi) OVCAR8 cells were more tumorigenic than SOX2(lo) cells when implanted in immunocompromised mice. "HIGH" gene expression (greater than mean fold increase) in these 3 markers demonstrated shorter progression-free survival compared with "LOW" expression: POSTN (5 vs 11 months, P = .02), ALDH1A1 (2 vs 11 months, P = .01), and SOX2 (6 vs 10 months, P = .04). Conclusions: Chemotherapy increased gene expression of 27 CSC markers in ovarian cancer. POSTN, ALDH1A1, and SOX2 significantly correlated with platinum resistance and higher expression predicted shorter progression-free survivals. The correlation of elevated CSC markers with poor prognosis highlights the need for the use of a CSC-directed agent to potentially extend survival of patients with ovarian cancer.

Although the role of the ErbB2/HER2 oncogene in cancers has been extensively studied, how ErbB2 is regulated remains poorly understood. A novel microRNA, mir-4728, was recently found within an intron of the ErbB2 gene. However, the function and clinical relevance of this intronic miRNA are completely unknown. Here, we demonstrate that mir-4728 is a negative regulator of MAPK signaling through directly targeting the ERK upstream kinase MST4 and exerts numerous tumor-suppressive properties in vitro and in animal models. Importantly, our patient sample study shows that mir-4728 was under-expressed in breast tumors compared with normal tissue, and loss of mir-4728 correlated with worse overall patient survival. These results strongly suggest that mir-4728 is a tumor-suppressive miRNA that controls MAPK signaling through targeting MST4, revealing mir-4728’s significance as a potential prognostic factor and target for therapeutic intervention in cancer. Moreover, this study represents a conceptual advance by providing strong evidence that a tumor-suppressive miRNA can antagonize the canonical signaling of its host oncogene.

Objectives: The cancer stem cell (CSC) theory of chemoresistance proposes that the proportion of CSCs correlate to enhanced chemoresistance and early disease recurrence. Therefore, novel therapeutics aimed at the innate molecular pathways responsible for this resistance would be paramount to overcoming platinum resistance in ovarian cancer.

Correction to: Cell Death and Differentiation (2012) 19, 378–386; doi:10.1038/cdd.2011.127; published online 7 October 2011 Since the publication of this article, the authors above have noticed that the column graphs of Figure 6e and f were incorrect in its online version. This error has now been rectified, and the correct article appears in this issue.

Objective: Cancer stem cells are considered to be primarily responsible for cancer self-renewal, invasion, and resistance to therapy. We describe a subpopulation of ovarian cancer cells with the surface marker profile CD44+/CD24– that exhibits the cancer stem cell properties of enhanced differentiation, invasion, resistance to therapy and correlation with survival.

Defensins play an important role in both innate and adaptive immunity due to their antimicrobial, regulatory, and chemotactic effects. Nonetheless, the role of murine β-defensins (mBD) 3 and 4, the murine homologs of human β-defensins (hBD) 2 and 3, remains unknown in Pseudomonas aeruginosa keratitis. This study explored their role in corneal infection and potential synergy with mBD2, a defensin associated with better outcome in this disease. Immunostaining and real-time RT-PCR data demonstrated that mBD3 and mBD4 expression was inducible and differentially regulated in the infected cornea of resistant BALB/c vs susceptible C57BL/6 (B6) mice. Knockdown studies using small interfering RNA treatment indicated that mBD3, but not mBD4, is required in ocular defense. Moreover, in vivo studies demonstrated individual and combined effects of mBD2 and mBD3 that modulate bacterial load, polymorphonuclear neutrophil (PMN) infiltration, and production of IFN-γ, MIP-2, IL-1β, TNF-α, inducible NO synthase (iNOS), TLR2, TLR4, MyD88, and NF-κB. Most notably, bacterial load was increased at 5 days postinfection by silencing either mBD2 or mBD3, but it was elevated at both 1 and 5 days postinfection when silencing both defensins. PMN infiltration was increased at 1 day postinfection by silencing both defensins or mBD3, but not mBD2 alone. iNOS expression was elevated by silencing mBD2, but it was reduced after silencing mBD3 or both defensins. Additionally, cell sources of mBD2 (macrophages, PMN and fibroblasts) and mBD3 (PMN) in corneal stroma were identified by dual label immunostaining after infection. Collectively, the data provide evidence that mBD2 and mBD3 together promote resistance against corneal infection.

Corneal infection with Pseudomonas aeruginosa results in corneal perforation in susceptible C57BL/6 (B6) mice, but not in resistant BALB/c mice. To explore the role of two important defensins, murine β-defensin-1 (mBD1) and mBD2, in the ocular immune defense system, their mRNA and protein expression levels were tested by real-time RT-PCR and Western blot, respectively. mRNA, protein, and immunostaining data demonstrated that both mBD1 and mBD2 were constitutively expressed in normal BALB/c and B6 corneas, and they were disparately up-regulated in BALB/c (more) vs B6 (less) corneas after infection. To determine whether either defensin played a role in host resistance, BALB/c mice were treated with either mBD1 or mBD2 small interfering RNA by subconjunctival injection together with topical application. Increased corneal opacity and worsened disease were displayed after knockdown of mBD2 but not of mBD1. mBD2 silencing also increased bacterial counts and polymorphonuclear neutrophil infiltration in BALB/c corneas. Real-time RT-PCR data further demonstrated that mBD2, not mBD1, differentially modulated mRNA expression of proinflammatory cytokines/molecules such as IFN-γ, MIP-2, IL-1β, TNF-α, IL-6, and inducible NO synthase; TLR signaling molecules, including TLR2, TLR4, TLR9, and MyD88; and the transcription factor NF-κB. Additionally, in vivo studies indicated that mBD2 silencing enhanced corneal nitrite levels and NF-κB activation. Collectively, the data provide evidence that mBD2, but not mBD1, is required for host resistance against P. aeruginosa-induced corneal infection.

Purpose: This study determined the role of inducible nitric oxide synthase ( iNOS) and nitric oxide ( NO) in the resistance response of BALB/c mice to P. aeruginosa-induced keratitis. Methods: RT-PCR, nitrite detection, iNOS inhibition, ELISA, and immunohistochemistry were used. Results: Early after infection, iNOS mRNA expression and nitrite levels in cornea were elevated compared to levels in the uninfected cornea. Treatment with aminoguanidine sulfate (AG), an inhibitor of iNOS, resulted in extensive corneal destruction, reduced nitrite levels, and reduced nitrotyrosine staining. Infected mice also had increased bacterial burden and elevated levels of MIP-1 alpha, IL-1 beta, and MIP-2 in the cornea. Dual-labeling immunohistochemistry established the macrophage as the major source of iNOS in the infected cornea. Conclusions: These data provide evidence that iNOS is constitutively expressed in the BALB/c cornea; that iNOS-derived NO is required for bacterial killing/stasis; and that the macrophage is the major cell source of NO.

Purpose. Antibody neutralization studies have shown that in Pseudomonas aeruginosa corneal infection, IL-1β is critical to regulation of the host inflammatory response, but mechanisms remain undetermined. To elucidate these mechanisms, caspase-1 knockout (ICE−/−) mice, that do not release mature IL-1β after endotoxin challenge, were tested. Methods. Clinical scores, MPO activity (for PMN quantitation), bacterial plate count, semiquantitative RT-PCR, ELISA and TUNEL staining were used to characterize the inflammatory response after infection in knockout and C57BL/6 (B6) wild type mice. Results. Clinical scores were significantly reduced in ICE−/− vs. B6 mice at 3, 5 and 7 days postinfection (p.i.). The decreased inflammatory response of ICE−/− mice was striking at 1 day p.i., and bacterial load also was significantly reduced in the cornea of the knockout mice at 3-7 days p.i. Knockout mice exhibited significantly increased mRNA and protein levels for IL-1Ra, the physiological regulator of IL-1 activity, and in addition, a significant increase in the number of apoptotic cells were quantitated in the corneal epithelium of ICE−/− vs. B6 mice at 1 day p.i. Conclusions. These data provide evidence that bacterial infection in the cornea of ICE−/− mice induces a reduced inflammatory response by: reduction in PMN and cytokines and chemokines that attract these cells to the cornea; enhanced apoptotic cell death in the infected epithelium; and increased IL-1Ra levels. The data also confirm the importance of IL-1 regulation in this model and suggest that ICE inhibition may be an attractive ancillary therapeutic strategy to control the host response to this pathogen.

Pseudomonas aeruginosa keratitis destroys the cornea in susceptible (B6), but not resistant (BALB/c) mice. To determine mechanisms mediating resistance, the role of IFN-γ, IL-12, and IL-18 was tested in BALB/c mice. RT-PCR analysis detected IFN-γ mRNA expression levels in cornea that were significantly increased at 1–7 days postinfection. IL-18 mRNA was detected constitutively in cornea and, at 1–7 days postinfection, levels were elevated significantly, while no IL-12 mRNA was similarly detected. To test whether IL-18 contributed to IFN-γ production, mice were treated with anti-IL-18 mAb. Treatment decreased corneal IFN-γ mRNA levels, and bacterial load and disease increased/worsened, compared with IgG-treated mice. To stringently examine the role of IFN-γ in bacterial killing, knockout (−/−) vs wild-type (wt) mice also were tested. All corneas perforated, and bacterial load was increased significantly in −/− vs wt mice. Because disease severity was increased in IFN-γ−/− vs IL-18-neutralized mice, and since IL-18 also induces production of TNF, we tested for TNF-α in both groups. ELISA analysis demonstrated significantly elevated corneal TNF-α protein levels in IFN-γ−/− vs wt mice after infection. In contrast, RT-PCR analysis of IL-18-neutralized vs IgG-treated infected mice revealed decreased corneal TNF-α mRNA expression. Next, to resolve whether TNF was required for bacterial killing, TNF-α was neutralized in BALB/c mice. No difference in corneal bacterial load was detected in neutralized vs IgG-treated mice. These data provide evidence that IL-18 contributes to the resistance response by induction of IFN-γ and that IFN-γ is required for bacterial killing.