Combinatorial host-response biomarker signature (BV score) and its subanalytes TRAIL, IP-10, and CRP in children with Mycoplasma pneumoniae community-acquired pneumonia.

BACKGROUND: Host-response biomarkers to differentiate bacterial from viral etiology in children with respiratory infections have shown high accuracies, but are understudied in Mycoplasma pneumoniae (Mp) infections. METHODS: We compared BV scores (0-34 indicating viral, and 66-100 indicating bacterial etiology), TRAIL (pg/mL), IP-10 (pg/mL), and CRP (mg/L) serum levels between Mp positive (Mp+) and negative (Mp-) community-acquired pneumonia (CAP). We performed receiver operating characteristic (ROC) curve analyses for clinical features and biomarkers. RESULTS: Of 80 CAP patients (median age 6.3 years, 57.5% male), 26 were Mp + CAP. By comparing Mp + CAP with Mp-CAP patients, BV scores were lower (median 14.0, IQR 3.0-27.8 vs. 54.0, IQR 12.0-84.8; P = 0.0008), TRAIL levels were higher (86.5, IQR 67.4-123.0 vs. 65.5, IQR 42.5-103.9; P = 0.025), CRP levels were lower (12.9, IQR 4.0-22.3 vs. 36.7, IQR 13.0-132.8; P = 0.0019), and IP-10 levels were comparable (366.0, IQR 150.2-603.8 vs. 331.0, IQR 154.3-878.8; P = 0.73). ROC analyses yielded a comparable discriminatory accuracy for the combination of age, fever duration, respiratory symptoms duration, with either procalcitonin or BV (AUC 0.87 vs. 0.86, P = 0.94). CONCLUSIONS: Children with Mp + CAP have atypically low, viral levels of the BV score, underscoring the complementary role of microbiological testing.

CRISPR activation screen identifies TGFß-associated PEG10 as a crucial tumor suppressor in Ewing sarcoma.

As the second most common pediatric bone and soft tissue tumor, Ewing sarcoma (ES) is an aggressive disease with a pathognomonic chromosomal translocation t(11;22) resulting in expression of EWS-FLI1, an "undruggable" fusion protein acting as transcriptional modulator. EWS-FLI1 rewires the protein expression in cancer cells by activating and repressing a multitude of genes. The role and contribution of most repressed genes remains unknown to date. To address this, we established a CRISPR activation system in clonal SKNMC cell lines and interrogated a custom focused library covering 871 genes repressed by EWS-FLI1. Among the hits several members of the TGFß pathway were identified, where PEG10 emerged as prime candidate due to its strong antiproliferative effect. Mechanistic investigations revealed that PEG10 overexpression caused cellular dropout via induction of cell death. Furthermore, non-canonical TGFß pathways such as RAF/MEK/ERK, MKK/JNK, MKK/P38, known to lead to apoptosis or autophagy, were highly activated upon PEG10 overexpression. Our study sheds new light onto the contribution of TGFß signalling pathway repression to ES tumorigenesis and suggest that its re-activation might constitute a novel therapeutic strategy.

CD4 + T cells are found within endemic Burkitt lymphoma and modulate Burkitt lymphoma precursor cell viability and expression of pathogenically relevant Epstein-Barr virus genes.

Endemic Burkitt lymphoma (eBL) is an aggressive B cell cancer characterized by an IgH/c-myc translocation and the harboring of Epstein-Barr virus (EBV). Evidence accumulates that CD4 + T cells might contribute to eBL pathogenesis. Here, we investigate the presence of CD4 + T cells in primary eBL tissue and their potential dichotomous impact on an EBV-infected pre-eBL cell model using ex vivo material and in vitro co-cultures. In addition, we establish a novel method to study the effect of IgH/c-myc translocation in primary B cells by employing a CRISPR/Cas9 knock-in approach to introduce and tag de novo translocation. We unprecedently document that CD4 + T cells are present in primary eBL tumor tissue. Furthermore, we demonstrate that CD4 + T cells on the one hand suppress eBL development by killing pre-eBL cells lacking IgH/c-myc translocation in vitro and on the other hand indirectly promote eBL development by inducing crucial EBV Latency III to Latency I switching in pre-eBL cells. Finally, we show that while the mere presence of an IgH/c-myc translocation does not suffice to escape CD4 + T-cell-mediated killing in vitro, the CD4 + T-cell-mediated suppression of EBV's Latency III program in vivo may allow cells harboring an IgH/c-myc translocation and additional mutations to evade immune control and proliferate by means of deregulated c-myc activity, resulting in neoplasia. Thus, our study highlights the dichotomous effects of CD4 + T cells and the mechanisms involved in eBL pathogenesis, suggests mechanisms of their impact on eBL progression, and provides a novel in vitro model for further investigation of IgH/c-myc translocation.

Support of BCP-ALL-cells by autologous bone marrow Th-cells involves induction of AID expression but not widespread AID off-target mutagenesis.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common childhood malignancy. The two-step BCP-ALL pathogenesis requires in utero-induced chromosomal aberrations and additional mutagenic events for overt leukemia. In mouse models, activation-induced cytidine deaminase (AID/AICDA) was suggested to contribute to BCP-ALL pathogenesis by off-target mutagenic activity. The role of AID in patients, however, remains unclear. Moreover, AID is usually not expressed in precursor B-cells but in germinal center B-cells, where it is induced upon T-helper (Th) cell stimulation. We have previously demonstrated that autologous Th-cells supportively interacted with BCP-ALL-cells. Here, we hypothesize that this interaction additionally induces AID expression in BCP-ALL-cells, leading to off-target mutagenic activity. We show that co-culture with autologous bone marrow Th-cells induced high AICDA expression in primary BCP-ALL-cells. This induction was mediated by a mechanism similar to the induction in mature B-cells involving IL-13/Stat6, CD40L/NF-κB and TGFß/Smad2/3 signaling. Even though Th-cell-induced AID seemed to be active in vitro in a BCP-ALL reporter cell line, extensive mutational signature analysis revealed no major contribution of AID activity to the mutational landscape in BCP-ALL patients. AID activity was neither detected in mutation clusters nor in known AID targets. Moreover, no recurrently mutated gene showed a relevant enrichment of mutations in the AID motif. Together, the lack of AID-induced mutational consequences argues towards a Th-cell-promoted yet AID-independent BCP-ALL pathogenesis and favors therapeutic research focusing on Th-cell-derived support of BCP-ALL-cells rather than AID-induced effects.