A novel experimental model of Cryptococcus neoformans-related immune reconstitution inflammatory syndrome (IRIS) provides insights into pathogenesis.

Antiretroviral therapy (ART) has yielded major advances in fighting the HIV pandemic by restoring protective immunity. However, a significant proportion of HIV patients co-infected with the opportunistic fungal pathogen Cryptococcus neoformans paradoxically develops a life-threatening immune reconstitution inflammatory syndrome (IRIS) during antiretroviral therapy. Despite several clinical studies, the underlying pathomecha-nisms are poorly understood. Here, we present the first mouse model of cryptococcal IRIS that allows for a detailed analysis of disease development. Lymphocyte-deficient RAG-1(-/-) mice are infected with C. neoformans and 4 weeks later adoptively transferred with purified CD4(+) T cells. Reconstitution of CD4(+) T cells is sufficient to induce a severe inflammatory disease similar to clinical IRIS in C. neoformans-infected RAG-1(-/-) mice of different genetic backgrounds and immunological phenotypes (i.e. C57BL/6 and BALB/c). Multiorgan inflammation is accompanied by a systemic release of distinct proinflammatory cytokines, i.e. IFN-γ, IL-6, and TNF-α. IRIS development is characterized by infection-dependent activation of donor CD4(+) T cells, which are the source of IFN-γ. Interestingly, IFN-γ-mediated effects are not required for disease induction. Taken together, this novel mouse model of cryptococcal IRIS provides a useful tool to verify potential mechanisms of pathogenesis, revealing targets for diagnosis and therapeutic interventions.

Abrogation of IL-4 receptor-α-dependent alternatively activated macrophages is sufficient to confer resistance against pulmonary cryptococcosis despite an ongoing T(h)2 response.

In the murine model of pulmonary infection with Cryptococcus neoformans, IL-4 receptor α (IL-4Rα)-dependent polyfunctional T(h)2 cells induce disease progression associated with alternative activation of lung macrophages. To characterize the effector role of IL-4Rα-dependent alternatively activated macrophages (aaMph), we intra-nasally infected mice with genetically ablated IL-4Rα expression on macrophages (LysM(Cre)IL-4Rα(-/lox) mice) and IL-4Rα(-/lox) littermates. LysM(Cre)IL-4Rα(-/lox) mice were significantly more resistant to pulmonary cryptococcosis with higher survival rates and lower lung burden than non-deficient heterozygous littermates. Infected LysM(Cre)IL-4Rα(-/lox) mice had reduced but detectable numbers of aaMph expressing arginase-1, chitinase-like enzyme (YM1) and CD206. Similar pulmonary expression of inducible nitric oxide synthase was found in LysM(Cre)IL-4Rα(-/lox) and IL-4Rα(-/lox) control mice, but macrophages from LysM(Cre)IL-4Rα(-/lox) mice showed a higher potential to produce nitric oxide. In contrast to the differences in the macrophage phenotype, pulmonary T(h)2 responses were similar in infected LysM(Cre)IL-4Rα(-/lox) and IL-4Rα(-/lox) mice with each mouse strain harboring polyfunctional T(h)2 cells. Consistently, type 2 pulmonary allergic inflammation associated with eosinophil recruitment and epithelial mucus production was present in lungs of both LysM(Cre)IL-4Rα(-/lox) and IL-4Rα(-/lox) mice. Our results demonstrate that, despite residual IL-4Rα-independent alternative macrophage activation and ongoing T(h)2-dependent allergic inflammation, abrogation of IL-4Rα-dependent aaMph is sufficient to confer resistance in pulmonary cryptococcosis. This is even evident on a relatively resistant heterozygous IL-4Rα(+/-) background indicating a key contribution of macrophage IL-4Rα expression to susceptibility in allergic bronchopulmonary mycosis.

Validation of the qSOFA and CRB-65 in SARS-CoV-2-infected community-acquired pneumonia.

Rationale: Prognostic accuracy of the quick sequential organ failure assessment (qSOFA) and CRB-65 (confusion, respiratory rate, blood pressure and age (≥65 years)) risk scores have not been widely evaluated in patients with SARS-CoV-2-positive compared to SARS-CoV-2-negative community-acquired pneumonia (CAP). The aim of the present study was to validate the qSOFA(-65) and CRB-65 scores in a large cohort of SARS-CoV-2-positive and SARS-CoV-2-negative CAP patients. Methods: We included all cases with CAP hospitalised in 2020 from the German nationwide mandatory quality assurance programme and compared cases with SARS-CoV-2 infection to cases without. We excluded cases with unclear SARS-CoV-2 infection state, transferred to another hospital or on mechanical ventilation during admission. Predefined outcomes were hospital mortality and need for mechanical ventilation. Results: Among 68 594 SARS-CoV-2-positive patients, hospital mortality (22.7%) and mechanical ventilation (14.9%) were significantly higher when compared to 167 880 SARS-CoV-2-negative patients (15.7% and 9.2%, respectively). All CRB-65 and qSOFA criteria were associated with both outcomes, and age dominated mortality prediction in SARS-CoV-2 (risk ratio >9). Scores including the age criterion had higher area under the curve (AUCs) for mortality in SARS-CoV-2-positive patients (e.g. CRB-65 AUC 0.76) compared to SARS-CoV-2 negative patients (AUC 0.68), and negative predictive value was highest for qSOFA-65=0 (98.2%). Sensitivity for mechanical ventilation prediction was poor with all scores (AUCs 0.59-0.62), and negative predictive values were insufficient (qSOFA-65=0 missed 1490 out of 10 198 patients (∼15%) with mechanical ventilation). Results were similar when excluding frail and palliative patients. Conclusions: Hospital mortality and mechanical ventilation rates were higher in SARS-CoV-2-positive than SARS-CoV-2-negative CAP. For SARS-CoV-2-positive CAP, the CRB-65 and qSOFA-65 scores showed adequate prediction of mortality but not of mechanical ventilation.

Eosinophils contribute to IL-4 production and shape the T-helper cytokine profile and inflammatory response in pulmonary cryptococcosis.

Susceptibility to infection with Cryptococcus neoformans is tightly determined by production of IL-4. In this study, we investigated the time course of IL-4 production and its innate cellular source in mice infected intranasally with C. neoformans. We show that pulmonary IL-4 production starts surprisingly late after 6 weeks of infection. Interestingly, in the lungs of infected mice, pulmonary T helper (Th) cells and eosinophils produce significant amounts of IL-4. In eosinophil-deficient ΔdblGATA mice, IL-33 receptor-expressing Th2s are significantly reduced, albeit not absent, whereas protective Th1 and Th17 responses are enhanced. In addition, recruitment of pulmonary inflammatory cells during infection with C. neoformans is reduced in the absence of eosinophils. These data expand previous findings emphasizing an exclusively destructive effector function by eosinophilic granulocytes. Moreover, in ΔdblGATA mice, fungal control is slightly enhanced in the lung; however, dissemination of Cryptococcus is not prevented. Therefore, eosinophils play an immunoregulatory role that contributes to Th2-dependent susceptibility in allergic inflammation during bronchopulmonary mycosis.

IL-4 receptor-alpha-dependent control of Cryptococcus neoformans in the early phase of pulmonary infection.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes lung inflammation and meningoencephalitis in immunocompromised people. Previously we showed that mice succumb to intranasal infection by induction of pulmonary interleukin (IL)-4Rα-dependent type 2 immune responses, whereas IL-12-dependent type 1 responses confer resistance. In the experiments presented here, IL-4Rα⁻/⁻ mice unexpectedly show decreased fungal control early upon infection with C. neoformans, whereas wild-type mice are able to control fungal growth accompanied by enhanced macrophage and dendritic cell recruitment to the site of infection. Lower pulmonary recruitment of macrophages and dendritic cells in IL-4Rα⁻/⁻ mice is associated with reduced pulmonary expression of CCL2 and CCL20 chemokines. Moreover, IFN-γ and nitric oxide production are diminished in IL-4Rα⁻/⁻ mice compared to wild-type mice. To directly study the potential mechanism(s) responsible for reduced production of IFN-γ, conventional dendritic cells were stimulated with C. neoformans in the presence of IL-4 which results in increased IL-12 production and reduced IL-10 production. Together, a beneficial role of early IL-4Rα signaling is demonstrated in pulmonary cryptococcosis, which contrasts with the well-known IL-4Rα-mediated detrimental effects in the late phase.

Migration of novel offset printing inks from cardboard packaging into food.

We report the migration potential of newly patented low-migration offset printing inks from cardboard food packaging and estimate the potential risk of their migration into food. The complete printing formulation was available and, due to the fact that the solvent compounds in these inks differ from those used in conventional printing inks, the investigation focused on these solvents. Instead of containing mineral and vegetable oils, the low-migration printing inks are based on a novel fatty acid ester. The migration of this alternative solvent was investigated according to DIN EN 14338 in Tenax simulant and in different types of food. For specific detection of the fatty acid ester, LC-MS/MS (APCI) was chosen due to its higher sensitivity and selectivity than GC/MS. Printed packaging materials from three different commercially available food products (meat, chocolate and sweets) were tested. Migration of the fatty acid ester from the packaging into simulants was analysed. For food samples, a clean-up method based on solid-phase extraction was developed and migration of the fatty acid ester into meat, chocolate and sweets was also demonstrated. Levels of contamination of these foods were between 5 and 80 microg fatty acid ester/kg, but levels in food were lower than those in simulants.