Better tools for better estimates: improving approaches to handling missing data in Swiss cancer registries.

OBJECTIVE: Various approaches have been used in the literature to handle missing vital status data in cancer registries. We aimed to compare these approaches to determine which led to the least biased estimates in typical analytic tasks of cancer registries. METHODS: A simulation study was performed using data from the Swiss National Agency for Cancer Registration for six tumor types. First, 5%, 10% and 15% missingness in the vital status were introduced artificially in the complete data. Second, missing vital status data were handled by applying no, single or multiple imputations. Five-year overall survival estimates, relative survival or standardized incidence ratio were computed. Estimates were compared with the true value. RESULTS: Standardized incidence ratio estimates for colorectal cancer obtained with multiple imputation yielded least biased results (-0.06 to -0.04), but the widest confidence intervals. Single imputation was more biased (-0.32) than using no imputation at all (-0.21). A similar pattern was observed for overall survival and relative survival. CONCLUSION: This simulation study indicated that often used single imputation (sometimes referred to as simulating follow-up times) techniques to fill in missing vital status data are likely too biased to be useful in practice. Multiple imputation approaches yielded standardized incidence ratio, overall and relative survival estimates with the least bias, indicating reasonable performance that is likely to generalize to other settings.

A vagal influence on schizophrenia? A nationwide retrospective cohort of vagotomized individuals.

Background and Objectives: Emerging preclinical evidence suggests that vagal signals contribute to the development of schizophrenia-related abnormalities in brain and behavior. Whether vagal communication in general, and its impairment in particular, is a risk factor for schizophrenia in humans remains, however, unclear. Vagotomy, the surgical lesion of the vagus nerve, was routinely performed as a treatment for peptic ulcer before modern treatment options were available. Hence, the primary aim of this study was to investigate whether vagotomy modulates the subsequent risk of developing schizophrenia. Moreover, given the existence of diverse vagotomy techniques (i.e., "truncal" or "selective"), our secondary goal was to test whether the extent of denervation modulates the risk of schizophrenia. Methods: Using a nationwide retrospective matched cohort design, we identified 8,315 vagotomized individuals from the Swedish National Patient Register during the period 1970-2020 and 40,855 non-vagotomized individuals matching for age, sex and type of peptic ulcer. The risk of being diagnosed with schizophrenia and associated psychoses (ICD10 codes F20-29) was analyzed using Cox proportional hazards regression models, including death as competing risk. Results: When considering all types of vagotomy together, vagotomy was not significantly associated with schizophrenia (HR: 0.91 [0.72; 1.16]). However, truncal vagotomy (which denervates all subdiaphragmatic organs) significantly increased the risk of developing schizophrenia by 69% (HR: 1.69 [1.08; 2.64]), whereas selective vagotomy (which only denervates the stomach) showed no significant association (HR: 0.80 [0.61; 1.04]). Discussion: Our results provide epidemiological support for the hypothesis that impairments in vagal functions could increase the risk of schizophrenia. Notably, the finding that truncal but not selective vagotomy is associated with an increased risk of schizophrenia raises the possibility that the activity of subdiaphragmatic non-gastric vagal branches may be of particular relevance for the development of schizophrenia.

'We are all in the same boat': a qualitative cross-sectional analysis of COVID-19 pandemic imagery in scientific literature and its use for people working in the German healthcare sector.

Background: The COVID-19 pandemic presents a significant challenge to professional responders in healthcare settings. This is reflected in the language used to describe the pandemic in the professional literature of the respective professions. The aim of this multidisciplinary study was to analyze the linguistic imagery in the relevant professional literature and to determine the identification of different professional groups with it and its emotional effects. Method: A list of 14 typical, widespread and differing imageries for COVID-19 in form of single sentences (e.g., "Until the pandemic is over, we can only run on sight.") were presented to 1,795 healthcare professionals in an online survey. The imageries had been extracted from a qualitative search in more than 3,500 international professional journals in medicine, psychology and theology. Ratings of agreement with these imageries and feelings about them were subjected to factor analysis. Results: Based on the list of imageries presented, it was possible to identify three factors for high/low agreement by experiences, and two factors for high/low induced feelings. Broad agreement emerged for imageries on "fight against the crisis" and "lessons learned from the crisis", while imageries on "acceptance of uncontrollability" tended to be rejected. Imageries of "challenges" tended to lead to a sense of empowerment among subjects, while imageries of "humility" tended to lead to a sense of helplessness. Conclusion: Based on the qualitative and subsequential quantitative analysis, several factors for imageries for the COVID-19 pandemic were identified that have been used in the literature. Agreement with imageries is mixed, as is the assessment of how helpful they are.

Cu-doped TiO2 nanoparticles improve local antitumor immune activation and optimize dendritic cell vaccine strategies.

Nanoparticle-mediated cancer immunotherapy holds great promise, but more efforts are needed to obtain nanoformulations that result in a full scale activation of innate and adaptive immune components that specifically target the tumors. We generated a series of copper-doped TiO2 nanoparticles in order to tune the kinetics and full extent of Cu2+ ion release from the remnant TiO2 nanocrystals. Fine-tuning nanoparticle properties resulted in a formulation of 33% Cu-doped TiO2 which enabled short-lived hyperactivation of dendritic cells and hereby promoted immunotherapy. The nanoparticles result in highly efficient activation of dendritic cells ex vivo, which upon transplantation in tumor bearing mice, exceeded the therapeutic outcomes obtained with classically stimulated dendritic cells. Efficacious but simple nanomaterials that can promote dendritic cancer cell vaccination strategies open up new avenues for improved immunotherapy and human health.

A TLR3 Ligand Reestablishes Chemotherapeutic Responses in the Context of FPR1 Deficiency.

For anthracycline-based chemotherapy to be immunogenic, dying cancer cells must release annexin A1 (ANXA1) that subsequently interacts with the pattern recognition receptor, formyl peptide receptor 1 (FPR1), on the surface of dendritic cells (DC). Approximately 30% of individuals bear loss-of-function alleles of FPR1, calling for strategies to ameliorate their anticancer immune response. Here, we show that immunotherapy with a ligand of Toll-like receptor-3, polyinosinic:polycytidylic acid (pIC), restores the deficient response to chemotherapy of tumors lacking ANXA1 developing in immunocompetent mice or those of normal cancers growing in FPR1-deficient mice. This effect was accompanied by improved DC- and T-lymphocyte-mediated anticancer immunity. Of note, carcinogen-induced breast cancers precociously developed in FPR1-deficient mice as compared with wild-type controls. A similar tendency for earlier cancer development was found in patients carrying the loss-of-function allele of FPR1. These findings have potential implications for the clinical management of FPR1-deficient patients. SIGNIFICANCE: The loss-of-function variant rs867228 in FPR1, harbored by approximately 30% of the world population, is associated with the precocious manifestation of breast, colorectal, esophageal, and head and neck carcinomas. pIC restores deficient chemotherapeutic responses in mice lacking Fpr1, suggesting a personalized strategy for compensating for the FPR1 defect.This article is highlighted in the In This Issue feature, p. 211.

The Dox-pDC - A murine conditionally immortalized plasmacytoid dendritic cell line with native immune profile.

Plasmacytoid dendritic cells (pDC) constitute a very rare blood cell population and play a significant role in immune response and immune-mediated disorders. Investigations on primary pDCs are hindered not only due to their rarity but also because they represent a heterogeneous cell population which is difficult to culture ex vivo. We generated a conditionally immortalized pDC line (Dox-pDC) from mice with Doxycycline-inducible SV40 Large T Antigen with a comparable immune profile to primary pDCs. The Dox-pDC secrete pro- and anti-inflammatory cytokines upon Toll-like receptor 9 stimulation and upregulate their MHCI, MHCII and costimulatory molecules. Further, the Dox-pDC activate and polarize naïve T cells in vivo and in vitro in response to the model antigen Ovalbumin. Due to their long-term culture stability and their robust proliferation Dox-pDC represent a reliable alternative to primary mouse pDC.

Neurotrophin Receptor p75NTR Regulates Immune Function of Plasmacytoid Dendritic Cells.

Plasmacytoid dendritic cells (pDCs) regulate innate and adaptive immunity. Neurotrophins and their receptors control the function of neuronal tissue. In addition, they have been demonstrated to be part of the immune response but little is known about the effector immune cells involved. We report, for the first time, the expression and immune-regulatory function of the low affinity neurotrophin receptor p75 neurotrophin receptor (p75NTR) by the antigen-presenting pDCs, mediated by toll-like receptor (TLR) 9 activation and differential phosphorylation of interferon regulatory factor 3 and 7. The modulation of p75NTR on pDCs significantly influences disease progression of asthma in an ovalbumin-induced mouse model mediated by the TLR9 signaling pathway. p75NTR activation of pDCs from patients with asthma increased allergen-specific T cell proliferation and cytokine secretion in nerve growth factor concentration-dependent manner. Further, p75NTR activation of pDCs delayed the onset of autoimmune diabetes in RIP-CD80GP mice and aggravated graft-versus-host disease in a xenotransplantation model. Thus, p75NTR signaling on pDCs constitutes a new and critical mechanism connecting neurotrophin signaling and immune response regulation with great therapeutic potential for a variety of immune disorders.

Defective IL-23/IL-17 Axis Protects p47phox-/- Mice from Colon Cancer.

In the colon, a sophisticated balance between immune reaction and tolerance is absolutely required. Dysfunction may lead to pathologic phenotypes ranging from chronic inflammatory processes to cancer development. Two prominent modulators of colon inflammation are represented by the closely related cytokines interleukin (IL)-12 and IL-23, which initiate adaptive Th1 and Th17 immune responses, respectively. In this study, we investigated the impact of the NADPH oxidase protein p47phox, which negatively regulates IL-12 in dendritic cells, on colon cancer development in a colitis-associated colon cancer model. Initially, we found that IL-12-/- mice developed less severe colitis but are highly susceptible to colon cancer. By contrast, p47phox-/- mice showed lower tumor scores and fewer high grade tumors than wild-type (WT) littermates. Treatment with toll-like receptor 9 ligand CpG2216 significantly enhanced colitis in p47phox-/- mice, whereas tumor growth was simultaneously reduced. In tumor tissue of p47phox-/- mice, the IL-23/IL-17 axis was crucially hampered. IL-23p19 protein expression in tumor tissue correlated with tumor stage. Reconstitution of WT mice with IL-23p19-/- bone marrow protected these mice from colon cancer, whereas transplantation of WT hematopoiesis into IL-23p19-/- mice increased the susceptibility to tumor growth. Our study strengthens the divergent role of IL-12 and IL-23 in colon cancer development. With the characterization of p47phox as a novel modulator of both cytokines our investigation introduces a promising new target for antitumor strategies.

Functional Restoration of gp91phox-Oxidase Activity by BAC Transgenesis and Gene Targeting in X-linked Chronic Granulomatous Disease iPSCs.

Chronic granulomatous disease (CGD) is an inherited immunodeficiency, caused by the inability of neutrophils to produce functional NADPH oxidase required for fighting microbial infections. The X-linked form of CGD (X-CGD), which is due to mutations in the CYBB (gp91phox) gene, a component of NADPH oxidase, accounts for about two-thirds of CGD cases. We derived induced pluripotent stem cells (iPSCs) from X-CGD patient keratinocytes using a Flp recombinase excisable lentiviral reprogramming vector. For restoring gp91phox function, we applied two strategies: transposon-mediated bacterial artificial chromosome (BAC) transgenesis and gene targeting using vectors with a fixed 5' homology arm (HA) of 8 kb and 3'HA varying in size from 30 to 80 kb. High efficiency of homologous recombination (up to 22%) was observed with increased size of the 3'HA. Both, BAC transgenesis and gene targeting resulted in functional restoration of the gp91phox measured by an oxidase activity assay in X-CGD iPSCs differentiated into the myeloid lineage. In conclusion, we delivered an important milestone towards the use of genetically corrected autologous cells for the treatment of X-CGD and monogenic diseases in general.

Generation of inducible immortalized dendritic cells with proper immune function in vitro and in vivo.

Dendritic cells are the professional antigen presenting cells of innate immunity and key players in maintaining the balance of immune responses. Studies with dendritic cells are mainly limited by their low numbers in vivo and their difficult maintenance in vitro. We differentiated bone marrow cells from transgenic mice expressing an inducible SV40 large T-antigen into dendritic cells. When immortalized by dexamethasone and doxycycline, these cells were stable in long-term culture. In the absence of dexamethasone and doxycycline (de-induction), dendritic cells displayed properties of primary cells, characterized by expression of classical dendritic cell surface markers CD11c, CD11b, MHCII, CD40 and CD86. Furthermore, de-induced lipopolysaccharide activated dendritic cells secreted IL-1ß, IL-6, TNFα and IL-12. De-induced, Ovalbumin-loaded dendritic cells polarize CD4(+) T cells into Th1, Th17 and Th2 cells, indicating their correct antigen presenting property. Consistent with intratracheal application of Ovalbumin-loaded primary dendritic cells into mice, the application of de-induced dendritic cells resulted in recruitment of lymphocytes to the lungs. In summary, we successfully expanded dendritic cells using conditional immortalization. The generated dendritic cells demonstrate the characteristic immunophenotype of primary dendritic cells and will facilitate further studies on immunomodulatory properties of dendritic cells.

CXCL9 causes heterologous desensitization of CXCL12-mediated memory T lymphocyte activation.

The chemokine receptors CXCR3 and CXCR4 are primarily involved in memory Th1 cell-driven autoimmune diseases. Although recent studies in chronic inflammatory disease showed therapeutic success using combined blockade, details of CXCR3 and CXCR4 synergism are not understood. In this investigation, we intended to unravel the interaction of these chemokine receptors in static and dynamic cell-migration assays at both the cellular and molecular levels. Effects of combined stimulation by murine CXCL9 and CXCL12, ligands of CXCR3 and CXCR4, respectively, were analyzed using a murine central memory Th1 cell clone. Costimulation with CXCL9 desensitized the chemotaxis of Th1 cells toward CXCL12 by up to 54%. This effect was found in murine EL-4 cells, as well as in primary human T cells. Furthermore, under dynamic flow conditions CXCL12-induced crawling and endothelial transmigration of Th1 cells was desensitized by CXCL9. Subsequent experiments uncovered several molecular mechanisms underlying the heterologous cross-regulation of CXCR4 signaling by the CXCR3 ligand. CXCR4 surface expression was reduced, whereas CXCL12-induced Akt phosphorylation and intracellular Ca(2+) signals were modulated. Moreover, blockade of Rac by NSC23766 revealed differential effects on CXCL12 and CXCL9 chemotaxis and abolished the desensitizing effect of CXCL9. The desensitization of CXCR4 via CXCR3 in memory Th1 cells suggests that their in vivo homeostasis, widely regulated by CXCL12, seemed to be significantly altered by CXCR3 ligands. Our data provide a more detailed understanding for the continuing extravasation and recruitment of Th1 lymphocytes into sites of persistent inflammation.

The histone demethylase UTX regulates stem cell migration and hematopoiesis.

Regulated migration of hematopoietic stem cells is fundamental for hematopoiesis. The molecular mechanisms underlying stem cell trafficking are poorly defined. Based on a short hairpin RNA library and stromal cell-derived factor-1 (SDF-1) migration screening assay, we identified the histone 3 lysine 27 demethylase UTX (Kdm6a) as a novel regulator for hematopoietic cell migration. Using hematopoietic stem and progenitor cells from our conditional UTX knockout (KO) mice, we were able to confirm the regulatory function of UTX on cell migration. Moreover, adult female conditional UTX KO mice displayed myelodysplasia and splenic erythropoiesis, whereas UTX KO males showed no phenotype. During development, all UTX KO female and a portion of UTX KO male embryos developed a cardiac defect, cranioschisis, and died in utero. Therefore, UTY, the male homolog of UTX, can compensate for UTX in adults and partially during development. Additionally, we found that UTX knockdown in zebrafish significantly impairs SDF-1/CXCR4-dependent migration of primordial germ cells. Our data suggest that UTX is a critical regulator for stem cell migration and hematopoiesis.

A rationally engineered anti-HIV peptide fusion inhibitor with greatly reduced immunogenicity.

Peptides derived from the C-terminal heptad repeat 2 (HR2) region of the HIV-1 gp41 envelope glycoprotein, so-called C peptides, are very efficient HIV-1 fusion inhibitors. We previously developed innovative gene therapeutic approaches aiming at the direct in vivo production of C peptides from genetically modified host cells and found that T cells expressing membrane-anchored or secreted C peptides are protected from HIV-1 infection. However, an unwanted immune response against such antiviral peptides may significantly impair clinical efficacy and pose safety risks to patients. To overcome this problem, we engineered a novel C peptide, V2o, with greatly reduced immunogenicity and excellent antiviral activity. V2o is based on the chimeric C peptide C46-EHO, which is derived from the HR2 regions of HIV-2(EHO) and HIV-1(HxB2) and has broad anti-HIV and anti-simian immunodeficiency virus activity. Antibody and major histocompatibility complex class I epitopes within the C46-EHO peptide sequence were identified by in silico and in vitro analyses. Using rational design, we removed these epitopes by amino acid substitutions and thus minimized antigenicity and immunogenicity considerably. At the same time, the antiviral activity of the deimmunized peptide V2o was preserved or even enhanced compared to that of the parental C46-EHO peptide. Thus, V2o is an ideal candidate, especially for those novel therapeutic approaches for HIV infection that involve direct in vivo production of antiviral C peptides.

The sphingosine kinase 1 and S1P1 axis specifically counteracts LPS-induced IL-12p70 production in immune cells of the spleen.

Sphingosine-1-phosphate (S1P) has been implicated in angiogenesis, inflammation, cancerogenesis, neurological excitability and immune regulation and is synthesized by two different sphingosine kinases (SphK). It was suggested that mice lacking the gene for SphK1 exhibit no obvious phenotype, because SphK2 compensates for its absence. However, recent investigations revealed that under challenge SphK1 contributed to pro-inflammatory processes favoring Th2 and Th17 rather than Th1-type reactions. To investigate the immune modulatory role of SphK1 as opposed to SphK2 specifically for the Th1 propagating IL-12p70 we compared WT and SphK1(-/-) splenocytes and Flt3-ligand differentiated BMCs of WT and SphK1(-/-), representing dendritic cells as major producers of IL-12p70, incubated with LPS. We determined the impact on IL-12p70 in comparison to other inflammatory cytokines, and on DC and macrophage surface marker expression, SphK mRNA, protein expression and enzymatic activity in splenocytes. Our data demonstrated that SphK1 deficiency enhanced LPS-induced IL-12p70 production although SphK2 was present. To further characterize SphK1-dependent IL-12p70 regulation we exogenously applied S1P, SEW2871 and the new potent S1P1 agonist CYM5442. Both S1P and S1P1-specific analogs fully compensated the increase of IL-12p70 production in SphK1-deficient splenocytes. The use of pertussis toxin, to block G(i)-coupled signaling downstream of S1P1, again increased IL-12p70 and neglected the compensation achieved by addition of S1P and S1P1 agonists pointing on the importance of this specific S1P-receptor. Given that, in parallel to a prominent IL-12p35 increase following LPS stimulation, LPS also enhanced SphK expression and total SphK activity, we concluded that SphK1-derived S1P acting via S1P1 is a major mechanism of this negative IL-12p70 feedback loop, which did not affect other cytokines. Moreover, our data showed that SphK2 activity failed to compensate for SphK1 deficiency. These findings clearly point to a divergent and cytokine-specific impact of immune cell SphK1 and SphK2 in chronic inflammation and cancer.

Ncf1 provides a reactive oxygen species-independent negative feedback regulation of TLR9-induced IL-12p70 in murine dendritic cells.

Permanent exposure to pathogens requires decisions toward tolerance or immunity as a prime task of dendritic cells. The molecular mechanisms preventing uncontrolled immune responses are not completely clear. We investigated the regulatory function of Ncf1, an organizing protein of NADPH oxidase, in the signaling cascade of Toll-like receptors. TLR9-stimulated spleen cells from both Ncf1-deficient and B10.Q mice with a point mutation in exon 8 of Ncf1 exhibited increased IL-12p70 secretion compared with controls. This finding was restricted to stimulatory CpG2216 and not induced by CpG2088. Because only CpG/TLR9-induced IL-12p70 was regulated by Ncf1, we used TRIF(-/-) and MyD88(-/-) cells to show that TLR9/MyD88 was primarily affected. Interestingly, additional experiments revealed that spleen cells from NOX2/gp91(phox)-deficient mice and the blocking of electron transfer by diphenylene iodonium had no influence on CpG-induced IL-12p70, confirming an NADPH oxidase-independent function of Ncf1. Finally, proving the in vivo relevance CpG adjuvant-guided OVA immunization resulted in a strong augmentation of IL-12p70-dependent Th1 IFN-gamma response only in Ncf1-deficient mice. These data suggest for the first time an important role for Ncf1 in the fine tuning of the TLR9/MyD88 pathway in vitro and in vivo that is independent of its role as an activator of NOX2.