UBQLN4 Represses Homologous Recombination and Is Overexpressed in Aggressive Tumors.

Genomic instability can be a hallmark of both human genetic disease and cancer. We identify a deleterious UBQLN4 mutation in families with an autosomal recessive syndrome reminiscent of genome instability disorders. UBQLN4 deficiency leads to increased sensitivity to genotoxic stress and delayed DNA double-strand break (DSB) repair. The proteasomal shuttle factor UBQLN4 is phosphorylated by ATM and interacts with ubiquitylated MRE11 to mediate early steps of homologous recombination-mediated DSB repair (HRR). Loss of UBQLN4 leads to chromatin retention of MRE11, promoting non-physiological HRR activity in vitro and in vivo. Conversely, UBQLN4 overexpression represses HRR and favors non-homologous end joining. Moreover, we find UBQLN4 overexpressed in aggressive tumors. In line with an HRR defect in these tumors, UBQLN4 overexpression is associated with PARP1 inhibitor sensitivity. UBQLN4 therefore curtails HRR activity through removal of MRE11 from damaged chromatin and thus offers a therapeutic window for PARP1 inhibitor treatment in UBQLN4-overexpressing tumors.

A Synergistic Interaction between Chk1- and MK2 Inhibitors in KRAS-Mutant Cancer.

KRAS is one of the most frequently mutated oncogenes in human cancer. Despite substantial efforts, no clinically applicable strategy has yet been developed to effectively treat KRAS-mutant tumors. Here, we perform a cell-line-based screen and identify strong synergistic interactions between cell-cycle checkpoint-abrogating Chk1- and MK2 inhibitors, specifically in KRAS- and BRAF-driven cells. Mechanistically, we show that KRAS-mutant cancer displays intrinsic genotoxic stress, leading to tonic Chk1- and MK2 activity. We demonstrate that simultaneous Chk1- and MK2 inhibition leads to mitotic catastrophe in KRAS-mutant cells. This actionable synergistic interaction is validated using xenograft models, as well as distinct Kras- or Braf-driven autochthonous murine cancer models. Lastly, we show that combined checkpoint inhibition induces apoptotic cell death in KRAS- or BRAF-mutant tumor cells directly isolated from patients. These results strongly recommend simultaneous Chk1- and MK2 inhibition as a therapeutic strategy for the treatment of KRAS- or BRAF-driven cancers.

Assessing the Influence of Sensor-Induced Noise on Machine-Learning-Based Changeover Detection in CNC Machines.

The noise in sensor data has a substantial impact on the reliability and accuracy of (ML) algorithms. A comprehensive framework is proposed to analyze the effects of diverse noise inputs in sensor data on the accuracy of ML models. Through extensive experimentation and evaluation, this research examines the resilience of a LightGBM ML model to ten different noise models, namely, Flicker, Impulse, Gaussian, Brown, Periodic, and others. A thorough analytical approach with various statistical metrics in a Monte Carlo simulation setting was followed. It was found that the Gaussian and Colored noise were detrimental when compared to Flicker and Brown, which are identified as safe noise categories. It was interesting to find a safe threshold limit of noise intensity for the case of Gaussian noise, which was missing in other noise types. This research work employed the use case of changeover detection in (CNC) manufacturing machines and the corresponding data from the publicly funded research project (OBerA).

Long-term analysis of the RiBVD phase II trial reveals the unfavorable impact of TP53 mutations and hypoalbuminemia in older adults with mantle cell lymphoma; for the LYSA group.

Between 2011 and 2012, a phase II trial evaluated the use of the RiBVD (Rituximab, Bendamustine, Velcade and Dexamethasone) combination as first-line treatment for mantle cell lymphoma (MCL) patients aged over 65. We have now re-examined the classic prognostic factors, adding an assessment of the mutation status of TP53. Patients (n=74; median age 73 years) were treated with the RiBVD combination. Median Progression Free Survival (mPFS) was 79 months, and median Overall Survival (mOS) was 111 months. TP53 mutation status was available for 54/74 (73%) patients. TP53 mutations (TP53mt) were found in 12 patients (22.2%). In multivariate analysis, among the prognostic factors (PF) evaluated, only TP53mt and an albumin level below 3.6 g/dL (Alb<3.6 g/dL) were independently associated with a shorter mPFS. A hazard ratio (HR) of 3.16 (1.3-9.9, p=0.014) was obtained for TP53mt versus TP53wt, and 3.6 (1.39-9.5, p=0.009) for Alb<3.6 g/dL vs Alb≥3.6 g/dL. In terms of mOS, multivariate analysis identified three PFs: TP53mt (HR: 5.9 (1.77-19.5, p=0.004)), Alb<3.6 g/dL (HR: 5.2 (1.46-18.5, p=0.011)), and ECOG=2 (HR: 3.7 (1.31-10.6, p=0.014)). Finally, a score combining TP53 status and albumin level distinguished three populations based on the presence of 0, 1, or 2 PF. For these populations, mPFS was 7.8 years, 28 months and 2.5 months, respectively. Our prolonged follow-up confirmed the efficacy of the RiBVD regimen, comparing it favorably to other regimens. TP53mt and hypoalbuminemia emerge as strong PF that can be easily integrated into prognostic scores for older adult patients with MCL.

Real-world study of the efficacy and safety of belantamab mafodotin (GSK2857916) in relapsed or refractory multiple myeloma based on data from the nominative ATU in France: the IFM 2020-04 study.

Belantamab mafodotin (BM) is an anti-BCMA antibody-drug conjugate (GSK2857916) that represents an alternative option in multiple myeloma. We sought to assess the efficacy and safety of BM in a real-world setting in patients who benefited from an early access program. We conducted an observational, retrospective, multicenter study. Eligibility criteria were treatment of relapsed or refractory multiple myeloma (RRMM) in monotherapy in adult patients who have received at least three lines of therapy previously, including at least one immunomodulatory agent (IMiD), a proteasome inhibitor (PI) and an anti-CD38 monoclonal antibody, and whose disease progressed during the last treatment period. The primary endpoint of the study is to assess the overall survival (OS). Between November 2019 and December 2020, 106 patients were treated with BM; 97 were eligible for the efficacy evaluation and 104 for safety. The median age was 66 (range, 37-82) years. High-risk cytogenetics were identified in 40.9% of patients. Fifty-five (56.7%) patients were triple-class refractory and 11 (11.3%) were penta-class refractory. The median number of prior lines of treatment was five (range, 3-12). The median number of BM cycles administered was three (range, 1-22). The overall response rate at best response was 38.1% (37/97). The median OS was 9.3 months (95% confidence interval [CI]: 5.9-15.3), and median progression-free survival was 3.5 months (95% CI: 1.9-4.7). The median duration of response was 9 months (range, 4.65-10.4). Treatment was delayed for 55 (52.9%) patients including 36.5% for treatment-related toxicity. Ophthalmic adverse events, mainly grade ≤2, were the most common toxicity (48%). The occurrence of keratopathy was 37.5%. Overall, our data are concordant with the results from DREAMM-2 in terms of efficacy and safety on a non-biased population.

A New Option for Pain Prevention Using a Therapeutic Virtual Reality Solution for Bone Marrow Biopsy (REVEH Trial): Open-Label, Randomized, Multicenter, Phase 3 Study.

BACKGROUND: Evidence regarding the analgesic effect of distraction through immersion in virtual reality (VR) for care-induced pain has been documented in several phase 2 trials, but comparison with standard treatments in large, randomized studies is needed. OBJECTIVE: In this open-label, multicenter, randomized, phase 3 trial, we evaluated the safety and efficacy of a novel VR therapy solution for distraction in the context of bone marrow biopsy. METHODS: Bliss is a VR software with 4 imaginary interactive environments in 3 dimensions with binaural sound (head-mounted display). Efficacy regarding pain intensity was evaluated using a visual analog scale (VAS; score from 0 to 10) immediately after the biopsy. Secondary end points were anxiety and tolerance. Modified intention-to-treat analysis was performed. RESULTS: Overall, 126 patients with previously documented untreated or suspected malignant hemopathy between September 6, 2018, and May 18, 2020, were randomly assigned in a 1:1 ratio to receive pain prevention with a mixture of nitrous oxide/oxygen (MEOPA; n=63) or VR (n=63) before and during the bone marrow biopsy. We excluded 8 patients from the final analysis (3 in the MEOPA group and 5 in the VR group). All patients received local anesthesia (lidocaine) before biopsy. Follow-up was limited to 1 month after the biopsy. Participants' median age was 65.5 (range 18-87) years, and 54.2% (64/118) of patients were male. The average pain intensity was 3.5 (SD 2.6, 95% CI -1.6 to 8.6) for the MEOPA group and 3.0 (SD 2.4, 95% CI -1.7 to 7.7) for the VR group, without any significant differences in age, sex, center, and hemopathy (P=.26). Concerning anxiety, 67.5% (79/117; fear of pain questionnaire) of the patients were afraid before the biopsy, and anxiety scores were moderate to very high in 26.3% (30/114; revised Spielberger State-Trait Anxiety Inventory questionnaire) of the patients before the biopsy and 9.0% (10/114) after the biopsy for all patients, without a significant difference between the 2 groups (P=.83). Immersion in VR was well tolerated by the majority (54/57, 95%) of patients in the VR group. CONCLUSIONS: The intensity of pain did not significantly differ between both arms. VR was well tolerated, and the satisfaction of patients, nurses, and physicians was very high. VR could be an alternative treatment in case of contraindication or intolerance to MEOPA. TRIAL REGISTRATION: ClinicalTrials.gov NCT03483194; https://clinicaltrials.gov/ct2/show/NCT03483194.

Powder Bed Monitoring Using Semantic Image Segmentation to Detect Failures during 3D Metal Printing.

Monitoring the metal Additive Manufacturing (AM) process is an important task within the scope of quality assurance. This article presents a method to gain insights into process quality by comparing the actual and target layers. Images of the powder bed were captured and segmented using an Xception-style neural network to predict the powder and part areas. The segmentation result of every layer is compared to the reference layer regarding the area, centroids, and normalized area difference of each part. To evaluate the method, a print job with three parts was chosen where one of them broke off and another one had thermal deformations. The calculated metrics are useful for detecting if a part is damaged or for identifying thermal distortions. The method introduced by this work can be used to monitor the metal AM process for quality assurance. Due to the limited camera resolutions and inconsistent lighting conditions, the approach has some limitations, which are discussed at the end.

Isolated intraocular relapses of primary cerebral lymphomas: An LOC network study.

Most relapses of primary central nervous system lymphoma (PCNSL) occur in the brain and are associated with a poor prognosis. Isolated intraocular relapses (IIORs) are rare and poorly described. We retrospectively selected from the French Lymphome Oculo-Cérébral database PCNSL patients who initially presented with cerebral localization and who experienced IIOR during the course of the disease. Of the 1472 patients included in the database, 55 patients presented an IIOR. Their median age was 68 years, and median Karnofsky Performance Status 80. IL-10 levels in the aqueous humor and/or in the vitreous were increased in 42/46 patients. 45/55 patients received systemic chemotherapy, and 11/55 received high-dose chemotherapy with autologous stem cell transplantation (HCT-ASCT) as consolidation treatment. After a median follow-up of 69 months, 42/55 patients had relapsed, including 90% of the patients who did not receive HCT-ASCT at IIOR and 40% of the patients who received HCT-ASCT at IIOR (p < 0.001). The first relapse after the initial IIOR was exclusively in the eye in 23/42 patients, and 29/42 patients had a subsequent brain relapse during the course of the disease. The median progression-free survival, brain-free survival and overall survival from IIOR were 12.2, 48.6 and 57.1 months, respectively. Isolated intraocular relapse is not exceptional in the course of PCNSL and deserves systematic ophthalmological follow-up. Its prognosis is much better than the prognosis of brain relapse, with an evolution close to that of primary vitreoretinal lymphoma. With the exception of patients who received HCT-ASCT at IIOR, almost all patients subsequently relapsed, often with other IIORs.

Evaluation of the Influence of Machine Tools on the Accuracy of Indoor Positioning Systems.

In recent years, the use of indoor localization techniques has increased significantly in a large number of areas, including industry and healthcare, primarily for monitoring and tracking reasons. From the field of radio frequency technologies, an ultra-wideband (UWB) system offers comparatively high accuracy and is therefore suitable for use cases with high precision requirements in position determination, for example for localizing an employee when interacting with a machine tool on the shopfloor. Indoor positioning systems with radio signals are influenced by environmental obstacles. Although the influence of building structures like walls and furniture was already analysed in the literature before, the influence of metal machine tools was not yet evaluated concerning the accuracy of the position determination. Accordingly, the research question for this article is defined: To what extent is the positioning accuracy of the UWB system influenced by a metal machine tool?The accuracy was measured in a test setup, which consists of a total of four scenarios in a production environment. For this purpose, the visual contact between the transmitter and the receiver modules, including the influence of further interfering factors of a commercially available indoor positioning system, was improved step by step from scenario 1 to 4. A laser tracker was used as the reference measuring device. The data was analysed based on the type A evaluation of standard uncertainty according to the guide to the expression of uncertainty in measurement (GUM). It was possible to show an improvement in standard deviation from 87.64cm±32.27cm to 6.07cm±2.24cm with confidence level 95% and thus provides conclusions about the setup of an indoor positioning system on the shopfloor.

Intravenous high-dose methotrexate based systemic therapy in the treatment of isolated primary vitreoretinal lymphoma: An LOC network study.

The treatment of primary vitreoretinal lymphoma (PVRL) remains controversial regarding the use of local, systemic, or combined treatments. The aim of this study was to analyze the efficacy and toxicity of intravenous high-dose methotrexate (IV HD-MTX) based systemic therapy in a uniformly treated population of PVRL patients. From a nationwide French database, we retrospectively selected 59 patients (median age: 70 years, median Karnofsky Performance Status: 90%) with isolated PVRL at diagnosis who received first-line treatment with HD-MTX between 2011 and 2018. 8/59 patients also received a local treatment. No deaths or premature discontinuations of MTX due to toxicity were reported. A complete response was obtained in 40/57 patients after chemotherapy. Before treatment, IL-10 was elevated in the aqueous humor (AH) or in the vitreous in 89% of patients. After treatment, AH IL-10 was undetectable in 87% of patients with a CR/uCR/PR and detectable in 92% of patients with PD/SD. After a median follow-up of 61 months, 42/59 (71%) patients had relapsed, including 29 isolated ocular relapses as the first relapse and a total of 22 brain relapses. The median overall survival, progression-free survival, ocular-free survival and brain-free survival were 75, 18, 29 and 73 months, respectively. IV HD-MTX based systemic therapy as a first-line treatment for isolated PVRL is feasible, with acceptable toxicity, even in an elderly population. This strategy seems efficient to prevent brain relapse with prolonged overall survival. However, the ocular relapse rate remains high. New approaches are needed to improve local control of this disease, and ocular assessment could be completed by monitoring AH IL-10.

Differential uptake of three clinically relevant allergens by human plasmacytoid dendritic cells.

BACKGROUND: Human plasmacytoid dendritic cells (pDC) have a dual role as interferon-producing and antigen-presenting cells. Their relevance for allergic diseases is controversial. and the impact of pDC on allergic immune responses is poorly understood. METHODS: This in vitro study on human pDC isolated from peripheral blood was designed to compare side by side the uptake of three clinically relevant representative allergens: fluorochrome-labeled house dust mite Der p 1, Bee venom extract from Apis mellifera (Api) and the food allergen OVA analyzed flow cytometry and confocal microscopy. RESULTS: We found that the internalization and its regulation by TLR9 ligation was significantly different between allergens in terms of time course and strength of uptake. Api and OVA uptake in pDC of healthy subjects was faster and reached higher levels than Der p 1 uptake. CpG ODN 2006 suppressed OVA uptake and to a lesser extent Der p 1, while Api internalization was not affected. All allergens colocalized with LAMP1 and EEA1, with Api being internalized particularly fast and reaching highest intracellular levels in pDC. Of note, we could not determine any specific differences in antigen uptake in allergic compared with healthy subjects. CONCLUSIONS: To our knowledge this is the first study that directly compares uptake regulation of clinically relevant inhalative, injective and food allergens in pDC. Our findings may help to explain differences in the onset and severity of allergic reactions as well as in the efficiency of AIT.

Tazemetostat for patients with relapsed or refractory follicular lymphoma: an open-label, single-arm, multicentre, phase 2 trial.

BACKGROUND: Activating mutations of EZH2, an epigenetic regulator, are present in approximately 20% of patients with follicular lymphoma. We investigated the activity and safety of tazemetostat, a first-in-class, oral EZH2 inhibitor, in patients with follicular lymphoma. METHODS: This study was an open-label, single-arm, phase 2 trial done at 38 clinics or hospitals in France, the UK, Australia, Canada, Poland, Italy, Ukraine, Germany, and the USA. Eligible patients were adults (≥18 years) with histologically confirmed follicular lymphoma (grade 1, 2, 3a, or 3b) that had relapsed or was refractory to two or more systemic therapies, had an Eastern Cooperative Oncology Group performance status of 0-2, and had sufficient tumour tissue for central testing of EZH2 mutation status. Patients were categorised by EZH2 status: mutant (EZH2mut) or wild-type (EZH2WT). Patients received 800 mg of tazemetostat orally twice per day in continuous 28-day cycles. The primary endpoint was objective response rate based on the 2007 International Working Group criteria for non-Hodgkin lymphoma, assessed by an independent radiology committee. Activity and safety analyses were done in patients who received one dose or more of tazemetostat. This study is registered with ClinicalTrials.gov, NCT01897571, and follow-up is ongoing. FINDINGS: Between July 9, 2015, and May 24, 2019, 99 patients (45 in the EZH2mut cohort and 54 in the EZH2WT cohort) were enrolled in the study. At data cutoff for the analysis (Aug 9, 2019), the median follow-up was 22·0 months (IQR 12·0-26·7) for the EZH2mut cohort and 35·9 months (24·9-40·5) for the EZH2WT cohort. The objective response rate was 69% (95% CI 53-82; 31 of 45 patients) in the EZH2mut cohort and 35% (23-49; 19 of 54 patients) in the EZH2WT cohort. Median duration of response was 10·9 months (95% CI 7·2-not estimable [NE]) in the EZH2mut cohort and 13·0 months (5·6-NE) in the EZH2WT cohort; median progression-free survival was 13·8 months (10·7-22·0) and 11·1 months (3·7-14·6). Among all 99 patients, treatment-related grade 3 or worse adverse events included thrombocytopenia (three [3%]), neutropenia (three [3%]), and anaemia (two [2%]). Serious treatment-related adverse events were reported in four (4%) of 99 patients. There were no treatment-related deaths. INTERPRETATION: Tazemetostat monotherapy showed clinically meaningful, durable responses and was generally well tolerated in heavily pretreated patients with relapsed or refractory follicular lymphoma. Tazemetostat is a novel treatment for patients with follicular lymphoma. FUNDING: Epizyme.

Performance of CT Compared with 18F-FDG PET in Predicting the Efficacy of Nivolumab in Relapsed or Refractory Hodgkin Lymphoma.

Background CT and fluorine 18 (18F) fluorodeoxyglucose (FDG) PET/CT performances following immune therapy are not well known in patients with relapsed or refractory Hodgkin lymphoma (RRHL). Purpose To compare CT and PET/CT for prognostic value of early response evaluation following nivolumab therapy. Materials and Methods This retrospective study included patients from 34 institutions who underwent early imaging response evaluation from July 2013 to April 2017. Three experienced readers classified imaging response by using Cheson et al and 2016 Lymphoma Response to Immunomodulatory Therapy Criteria as follows: complete (metabolic) response, partial (metabolic) response, stable disease or no metabolic response, or progressive (metabolic) disease. Primary CT and PET assessments were performed at a median of 2.0 months (interquartile range, 1.7-3.7 months) after nivolumab initiation. Kaplan-Meier analysis was used to determine the relationship of primary CT and PET assessment response categories to overall survival (OS). Agreements between primary and secondary imaging assessments were assessed by using κ analysis. Results A total of 45 patients (median age, 37 years; range, 18-77 years; 25 men) underwent a primary assessment using CT and PET/CT; 36 patients also underwent a subsequent assessment. Eleven patients (24%) died after a median follow-up of 21.2 months. CT and PET response categories were associated with OS (P = .03 for primary CT assessment; P = .02 for primary PET assessment). There was no pseudoprogression at primary CT and PET assessments. At the primary assessment, response categories by using CT were reclassified by using PET in 44% (20 of 45) of patients. Among these, 55% (11 of 20) were reclassified to complete metabolic response (complete metabolic response rate: 29% [13 of 45 patients] vs complete response rate: 4% [two of 45 patients]), with a 2-year OS probability of 100%. At the secondary assessment, complete response rate using CT increased to 17% (six of 36 patients), hence a better agreement with PET (κ = 0.78; P < .001). Conclusion Early CT and PET/CT at a median of 2 months after initiation of nivolumab predicted overall survival in relapsed or refractory Hodgkin lymphoma. Early PET detected additional patients with complete metabolic response. © RSNA, 2020 Online supplemental material is available for this article. See also the editorial by Scott and Wang in this issue.

A phase 2 study of rituximab, bendamustine, bortezomib and dexamethasone for first-line treatment of older patients with mantle cell lymphoma.

We present results of a prospective, multicenter, phase II study evaluating rituximab, bendamustine, bortezomib and dexamethasone as first-line treatment for patients with mantle cell lymphoma aged 65 years or older. A total of 74 patients were enrolled (median age, 73 years). Patients received a maximum of six cycles of treatment at 28-day intervals. The primary objective was to achieve an 18-month progression-free survival rate of 65% or higher. Secondary objectives were to evaluate toxicity and the prognostic impact of mantle cell lymphoma prognostic index, Ki67 expression, [18F]fluorodeoxyglucose-positron emission tomography and molecular minimal residual disease, in peripheral blood or bone marrow. With a median follow-up of 52 months, the 24-month progression-free survival rate was 70%, hence the primary objective was reached. After six cycles of treatment, 91% (54/59) of responding patients were analyzed for peripheral blood residual disease and 87% of these (47/54) were negative. Four-year overall survival rates of the patients who did not have or had detectable molecular residual disease in the blood at completion of treatment were 86.6% and 28.6%, respectively (P<0.0001). Neither the mantle cell lymphoma index, nor fluorodeoxyglucose-positron emission tomography nor Ki67 positivity (cut off of ≥30%) showed a prognostic impact for survival. Hematologic grade 3-4 toxicities were mainly neutropenia (51%), thrombocytopenia (35%) and lymphopenia (65%). Grade 3-4 non-hematologic toxicities were mainly fatigue (18.5%), neuropathy (15%) and infections. In conclusion, the tested treatment regimen is active as frontline therapy in older patients with mantle cell lymphoma, with manageable toxicity. Minimal residual disease status after induction could serve as an early predictor of survival in mantle cell lymphoma. ClinicalTrials.gov: NCT 01457144.

DNA damage pathways and B-cell lymphomagenesis.

PURPOSE OF REVIEW: Recent lymphoma genome sequencing projects have shed light on the genomic landscape of indolent and aggressive lymphomas, as well as some of the molecular mechanisms underlying recurrent mutations and translocations in these entities. Here, we review these recent genomic discoveries, focusing on acquired DNA repair defects in lymphoma. In addition, we highlight recently identified actionable molecular vulnerabilities associated with recurrent mutations in chronic lymphocytic leukemia (CLL), which serves as a model entity. RECENT FINDINGS: The results of several large lymphoma genome sequencing projects have recently been reported, including CLL, T-PLL and DLBCL. We align these discoveries with proposed mechanisms of mutation acquisition in B-cell lymphomas. Moreover, novel autochthonous mouse models of CLL have recently been generated and we discuss how these models serve as preclinical tools to drive the development of novel targeted therapeutic interventions. Lastly, we highlight the results of early clinical data on novel compounds targeting defects in the DNA damage response of CLL with a particular focus on deleterious ATM mutations. SUMMARY: Defects in DNA repair pathways are selected events in cancer, including lymphomas. Specifically, ATM deficiency is associated with PARP1- and DNA-PKcs inhibitor sensitivity in vitro and in vivo.

Human NK cells adapt their immune response towards increasing multiplicities of infection of Aspergillus fumigatus.

BACKGROUND: The saprophytic fungus Aspergillus fumigatus reproduces by generation of conidia, which are spread by airflow throughout nature. Since humans are inhaling certain amounts of spores every day, the (innate) immune system is constantly challenged. Even though macrophages and neutrophils carry the main burden, also NK cells are regarded to contribute to the antifungal immune response. While NK cells reveal a low frequency, expression and release of immunomodulatory molecules seem to be a natural way of their involvement. RESULTS: In this study we show, that NK cells secrete chemokines such as CCL3/MIP-1α, CCL4/MIP-1ß and CCL5/RANTES early on after stimulation with Aspergillus fumigatus and, in addition, adjust the concentration of chemokines released to the multiplicity of infection of Aspergillus fumigatus. CONCLUSIONS: These results further corroborate the relevance of NK cells within the antifungal immune response, which is regarded to be more and more important in the development and outcome of invasive aspergillosis in immunocompromised patients after hematopoietic stem cell transplantation. Additionally, the correlation between the multiplicity of infection and the expression and release of chemokines shown here may be useful in further studies for the quantification and/or surveillance of the NK cell involvement in antifungal immune responses.

Validation of a simplified in vitro Transwell® model of the alveolar surface to assess host immunity induced by different morphotypes of Aspergillus fumigatus.

Interactions between fungal pathogens such as Aspergillus fumigatus with host alveolar epithelium and innate immune cells are crucial in the defense against opportunistic fungal infections. In this study a simplified Transwell® system with a confluent layer of A549 cells acted as a model for the alveolar surface. A. fumigatus and dendritic cells were added to simulate the spatial and cellular complexity in the alveolus. Fungal growth into the lower chamber was validated by galactomannan assays. Addition of moDCs to the upper chamber led to a reduced GM signal and fungal growth, indicating moDC antifungal activity. Minimal cell death was documented by analyses of lactate dehydrogenase concentrations and pro-apoptotic gene expression. Measurement of trans-epithelial dextran blue movement confirmed tightness of the epithelial barrier even in presence of A. fumigatus. Cytokine measurements in supernatants from both chambers of the Transwell® system documented distinct response patterns during early and late stages of epithelial invasion, with A549 cells appearing to make a minimal contribution to cytokine release. Concentrations of cytokines in the lower chamber varied distinctly from the upper chamber, depending on the molecular weight of the cytokines. Low inter-assay variability of fungal biomarkers and cytokines was confirmed, highlighting that in vitro models closely mimicking conditions in the human lung can facilitate reproducible measurement of the dynamics of cytokine release and fungal penetration of host epithelia.

Human primary myeloid dendritic cells interact with the opportunistic fungal pathogen Aspergillus fumigatus via the C-type lectin receptor Dectin-1.

Aspergillus fumigatus is an opportunistic fungal pathogen causing detrimental infections in immunocompromised individuals. Dendritic cells (DCs) are potent antigen-presenting cells and recognize the A. fumigatus cell wall component ß-1,3 glucan via Dectin-1, followed by DC maturation and cytokine release. Here, we demonstrate that human primary myeloid DCs (mDCs) interact with different morphotypes of A. fumigatus. Dectin-1 is expressed on mDCs and is down-regulated after contact with A. fumigatus, indicating that mDCs recognize A. fumigatus via this receptor. Blocking of Dectin-1, followed by stimulation with depleted zymosan diminished the up-regulation of the T-cell co-stimulatory molecules CD40, CD80, HLA-DR and CCR7 on mDCs and led to decreased release of the cytokines TNF-α, IL-8, IL-1ß and IL-10.

Human Invariant Natural Killer T cells possess immune-modulating functions during Aspergillus infection.

Aspergillus fumigatus is the most common cause for invasive fungal infections, a disease associated with high mortality in immune-compromised patients. CD1d-restricted invariant natural killer T (iNKT) cells compose a small subset of T cells known to impact the immune response toward various infectious pathogens. To investigate the role of human iNKT cells during A. fumigatus infection, we studied their activation as determined by CD69 expression and cytokine production in response to distinct fungal morphotypes in the presence of different CD1d(+) antigen presenting cells using flow cytometry and multiplex enzyme-linked immunosorbent assay (ELISA). Among CD1d(+) subpopulations, CD1d(+)CD1c(+) mDCs showed the highest potential to activate iNKT cells on a per cell basis. The presence of A. fumigatus decreased this effect of CD1d(+)CD1c(+) mDCs on iNKT cells and led to reduced secretion of TNF-α, G-CSF and RANTES. Production of other Th1 and Th2 cytokines was not affected by the fungus, suggesting an immune-modulating function for human iNKT cells during A. fumigatus infection.