Glycosylation of Candida albicans cell wall proteins is critical for induction of innate immune responses and apoptosis of epithelial cells.

C. albicans is one of the most common fungal pathogen of humans, causing local and superficial mucosal infections in immunocompromised individuals. Given that the key structure mediating host-C. albicans interactions is the fungal cell wall, we aimed to identify features of the cell wall inducing epithelial responses and be associated with fungal pathogenesis. We demonstrate here the importance of cell wall protein glycosylation in epithelial immune activation with a predominant role for the highly branched N-glycosylation residues. Moreover, these glycan moieties induce growth arrest and apoptosis of epithelial cells. Using an in vitro model of oral candidosis we demonstrate, that apoptosis induction by C. albicans wild-type occurs in early stage of infection and strongly depends on intact cell wall protein glycosylation. These novel findings demonstrate that glycosylation of the C. albicans cell wall proteins appears essential for modulation of epithelial immunity and apoptosis induction, both of which may promote fungal pathogenesis in vivo

High Biofilm Formation of Non-Smooth Candida parapsilosis Correlates with Increased Incorporation of GPI-Modified Wall Adhesins

Candida parapsilosis is among the most frequent causes of candidiasis. Clinical isolates of this species show large variations in colony morphotype, ranging from round and smooth to a variety of non-smooth irregular colony shapes. A non-smooth appearance is related to increased formation of pseudohyphae, higher capacity to form biofilms on abiotic surfaces, and invading agar. Here, we present a comprehensive study of the cell wall proteome of C. parapsilosis reference strain CDC317 and seven clinical isolates under planktonic and sessile conditions. This analysis resulted in the identification of 40 wall proteins, most of them homologs of known Candida albicans cell wall proteins, such as Gas, Crh, Bgl2, Cht2, Ecm33, Sap, Sod, Plb, Pir, Pga30, Pga59, and adhesin family members. Comparative analysis of exponentially growing and stationary phase planktonic cultures of CDC317 at 30 °C and 37 °C revealed only minor variations. However, comparison of smooth isolates to non-smooth isolates with high biofilm formation capacity showed an increase in abundance and diversity of putative wall adhesins from Als, Iff/Hyr, and Hwp families in the latter. This difference depended more strongly on strain phenotype than on the growth conditions, as it was observed in planktonic as well as biofilm cells. Thus, in the set of isolates analyzed, the high biofilm formation capacity of non-smooth C. parapsilosis isolates with elongated cellular phenotypes correlates with the increased surface expression of putative wall adhesins in accordance with their proposed cellular function.This work was funded by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) (SAF2013-47570-P and SAF2017-86188-P) and the regional government of Castilla-La Mancha (JCCM) (SBPLY/19/180501/000114), all co-financed by the EU (FEDER), to P.W.J.d.G. and E.E., the Consejería de Educación, Universidades e Investigación (GIC15/78 IT-990-16) of Gobierno Vasco-Eusko Jaurlaritza to E.E., and the FP7-PEOPLE-2013-ITN—Marie-Curie Action: “Initial Training Networks”: Molecular Mechanisms of Human Fungal Pathogen Host Interaction, ImResFun, MC-ITN-606786, to O.B

WWP2 ubiquitylates RNA polymerase II for DNA-PK-dependent transcription arrest and repair at DNA breaks

DNA double-strand breaks (DSBs) at RNA polymerase II (RNAPII) transcribed genes lead to inhibition of transcription. The DNA-dependent protein kinase (DNA-PK) complex plays a pivotal role in transcription inhibition at DSBs by stimulating proteasome-dependent eviction of RNAPII at these lesions. How DNA-PK triggers RNAPII eviction to inhibit transcription at DSBs remains unclear. Here we show that the HECT E3 ubiquitin ligase WWP2 associates with components of the DNA-PK and RNAPII complexes and is recruited to DSBs at RNAPII transcribed genes. In response to DSBs, WWP2 targets the RNAPII subunit RPB1 for K48-linked ubiquitylation, thereby driving DNA-PK- and proteasome-dependent eviction of RNAPII. The lack of WWP2 or expression of nonubiquitylatable RPB1 abrogates the binding of nonhomologous end joining (NHEJ) factors, including DNA-PK and XRCC4/DNA ligase IV, and impairs DSB repair. These findings suggest that WWP2 operates in a DNA-PK-dependent shutoff circuitry for RNAPII clearance that promotes DSB repair by protecting the NHEJ machinery from collision with the transcription machinery

Risk factors for otitis media in children with special emphasis on the role of colonization with bacterial airway pathogens: the Generation R study

Acute otitis media is the most frequent diagnosis in children visiting physicians’ offices. Risk factors for otitis media have been widely studied. Yet, the correlation between bacterial carriage and the development of otitis media is not entirely clear. Our aim was to study in a population-based prospective cohort the risk factors for otitis media in the second year of life with special emphasis on the role of colonization with Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis. The study was embedded in the Generation R Study. Data on risk factors and doctor-diagnosed otitis media were obtained by midwives, hospital registries and postal questionnaires in the whole cohort (n = 7,295). Nasopharyngeal swabs were obtained at the age of 1.5, 6 and 14 months in the focus cohort (n = 1,079). Of these children, 2,515 (47.2%) suffered at least one period of otitis media in their second year of life. The occurrence of otitis media during the follow-up period in the first 6 months of life and between 6 and 12 months of age was associated with the risk of otitis media in the second year of life (aOR, 1.83 95% CI 1.24–2.71 and aOR 2.72, 95% CI 2.18–3.38, respectively). Having siblings was associated with an increased risk for otitis media in the second year of life (aOR 1.42, 95% CI 1.13–1.79). No associations were found between bacterial carriage in the first year of life and otitis media in the second year of life. In our study, otitis media in the first year of life is an independent risk factor for otitis media in the second year of life. Surprisingly, bacterial carriage in the first year of life did not add to this risk. Moreover, no association was observed between bacterial carriage in the first year of life and otitis in the second year of life

Healthcare Costs of Metastatic Cutaneous Melanoma in the Era of Immunotherapeutic and Targeted Drugs

Immunotherapeutic and targeted drugs improved survival of patients with metastatic melanoma. There is, however, a lack of evidence regarding their healthcare costs in clinical practice. The aim of our study was to provide insight into real-world healthcare costs of patients with metastatic cutaneous melanoma. Data were obtained from the Dutch Melanoma Treatment Registry for patients who were registered between July 2012 and December 2018. Mean total/monthly costs per patient were reported for all patients, patients who did not receive systemic therapy, and patients who received systemic therapy. Furthermore, mean episode/monthly costs per line of therapy and drug were reported for patients who received systemic therapy. Mean total/monthly costs were € 89,240/€ 6809: € 7988/€ 2483 for patients who did not receive systemic therapy (n = 784) and € 105,078/€ 7652 for patients who received systemic therapy (n = 4022). Mean episode/monthly costs were the highest for nivolumab plus ipilimumab (€ 79,675/€ 16,976), ipilimumab monotherapy (€ 79,110/€ 17,252), and dabrafenib plus trametinib (€ 77,053/€ 12,015). Dacarbazine yielded the lowest mean episode/monthly costs (€ 6564/€ 2027). Our study showed that immunotherapeutic and targeted drugs had a large impact on real-world healthcare costs. As new drugs continue entering the treatment landscape for (metastatic) melanoma, it remains crucial to monitor whether the benefits of these drugs outweigh their costs

Discontinuation of anti-PD-1 monotherapy in advanced melanoma:Outcomes of daily clinical practice

There is no consensus on the optimal treatment duration of anti-PD-1 for advanced melanoma. The aim of our study was to gain insight into the outcomes of anti-PD-1 discontinuation, the association of treatment duration with progression and anti-PD-1 re-treatment in relapsing patients. Analyses were performed on advanced melanoma patients in the Netherlands who discontinued first-line anti-PD-1 monotherapy in the absence of progressive disease (n = 324). Survival was estimated after anti-PD-1 discontinuation and with a Cox model the association of treatment duration with progression was assessed. At the time of anti-PD-1 discontinuation, 90 (28%) patients had a complete response (CR), 190 (59%) a partial response (PR) and 44 (14%) stable disease (SD). Median treatment duration for patients with CR, PR and SD was 11.2, 11.5 and 7.2 months, respectively. The 24-month progression-free survival and overall survival probabilities for patients with a CR, PR and SD were, respectively, 64% and 88%, 53% and 82%, 31% and 64%. Survival outcomes of patients with a PR and CR were similar when anti-PD-1 discontinuation was not due to adverse events. Having a PR at anti-PD-1 discontinuation and longer time to first response were associated with progression [hazard ratio (HR) = 1.81 (95% confidence interval, CI = 1.11-2.97) and HR = 1.10 (95% CI = 1.02-1.19; per month increase)]. In 17 of the 27 anti-PD-1 re-treated patients (63%), a response was observed. Advanced melanoma patients can have durable remissions after (elective) anti-PD-1 discontinuation

Survival outcomes of patients with advanced mucosal melanoma diagnosed from 2013 to 2017 in the Netherlands - A nationwide population-based study

Background: Mucosal melanoma (MM) is rare and has a poor prognosis. Since 2011, new effective treatments are available for advanced melanoma. It is unclear whether patients with mucosal melanoma equally benefit from these new treatments compared with patients with cutaneous melanoma (CM). Methods: Patients with advanced MM and CM diagnosed between 2013 and 2017 were included from a nationwide population-based registry – the Dutch Melanoma Treatment Registry. Overall survival (OS) was estimated with the Kaplan-Meier method (also for a propensity score-matched cohort). A Cox model was used to analyse the association of possible prognostic factors with OS. Results: In total, 120 patients with MM and 2960 patients with CM were included. Median OS was 8.7 months and 14.5 months, respectively. Patients with MM were older (median age 70 versus 65 years) and more often female (60% versus 41%), compared with CM. In total, 77% and 2% of the MM patients were treated with first-line immunotherapy and targeted therapy, respectively, compared with 49% and 33% of the CM patients. In contrast to CM, OS for MM did not improve for patients diagnosed in 2015–2017, compared with 2013–2014. ECOG performance score ≥1 (HR = 1.99 [1.26–3.15; p = 0.003]) and elevated LDH level (HR = 1.63 [0.96–2.76]; p = 0.069) in MM were associated with worse survival. Conclusions: Within the era of immune and targeted therapies, prognosis for patients with advanced MM has not improved as much as for CM. Collaboration is necessary to enlarge sample size for research to improve immunotherapeutic strategies and identify targetable mutations

Checkpoint inhibitor induced hepatitis and the relation with liver metastasis and outcome in advanced melanoma patients

BACKGROUND: Checkpoint inhibitor-induced hepatitis is an immune-related adverse event of programmed cell death protein 1 (PD-1) inhibition, cytotoxic T-lymphocyte associated 4 (CTLA-4) inhibition or the combination of both. Aim of this study was to assess whether checkpoint inhibitor-induced hepatitis is related to liver metastasis and outcome in a real-world nationwide cohort. METHODS: Data from the prospective nationwide Dutch Melanoma Treatment Registry (DMTR) was used to analyze incidence, risk factors of checkpoint inhibitor-induced grade 3–4 hepatitis and outcome. RESULTS: 2561 advanced cutaneous melanoma patients received 3111 treatments with checkpoint inhibitors between May 2012 and January 2019. Severe hepatitis occurred in 30/1620 (1.8%) patients treated with PD-1 inhibitors, in 29/1105 (2.6%) patients treated with ipilimumab and in 80/386 (20.7%) patients treated with combination therapy. Patients with hepatitis had a similar prevalence of liver metastasis compared to patients without hepatitis (32% vs. 27%; p = 0.58 for PD-1 inhibitors; 42% vs. 29%; p = 0.16 for ipilimumab; 38% vs. 43%; p = 0.50 for combination therapy). There was no difference in median progression free and overall survival between patients with and without hepatitis (6.0 months vs. 5.4 months progression-free survival; p = 0.61; 17.0 vs. 16.2 months overall survival; p = 0.44). CONCLUSION: Incidence of hepatitis in a real-world cohort is 1.8% for PD-1 inhibitor, 2.6% for ipilimumab and 20.7% for combination therapy. Checkpoint inhibitor-induced hepatitis had no relation with liver metastasis and had no negative effect on the outcome. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12072-021-10151-4