Ali, Mohamed F.

Abstract Purpose: Monitoring disease progression in patients with high-grade gliomas (HGG) is challenging due to treatment-related changes in imaging and the requirement for neurosurgical intervention to obtain diagnostic tissue. DNA junctions in HGG often amplify oncogenes, making these DNA fragments potentially more abundant in blood than monoallelic mutations. In this study, we piloted a cell-free DNA approach for disease detection in the plasma of patients with HGG by leveraging patient-specific DNA junctions associated with oncogene amplifications. Experimental Design: Whole-genome sequencing of grade 3 or 4 isocitrate dehydrogenase–mutant or wild-type astrocytomas was utilized to identify amplified junctions. Individualized qPCR assays were developed using patient-specific primers designed for the amplified junction. ctDNA levels containing these junctions were measured in patient plasma samples. Results: Unique amplified junctions were evaluated by individualized semi-qPCR assays in presurgical plasma of 18 patients, 15 with tumor-associated focal amplifications and three without tumor-associated focal amplifications. high copy-number junctions were robustly detected in the plasma of 14 of 15 (93.3%) patients with amplified junctions and none of the controls. Changes in junction abundance correlated with disease trajectory in serial plasma samples from five patients, including increased abundance of amplified junctions preceding radiographic disease progression. Conclusions: In patients with grade 3 or 4 astrocytomas who had tumor-associated amplifications, patient-specific amplified junctions were successfully detected in assayed plasma from most patients. Longitudinal analysis of plasma samples correlated with disease trajectory, including cytoreduction and progression.

Abstract B-cell activation is increasingly linked to numerous fibrotic lung diseases, and it is well known that aggregates of lymphocytes form in the lung of many of these patients. Activation of B-cells by pattern recognition receptors (PRRs) drives the release of inflammatory cytokines, chemokines, and metalloproteases important in the pathophysiology of pulmonary fibrosis. However, the specific mechanisms of B-cell activation in patients with idiopathic pulmonary fibrosis (IPF) are poorly understood. Herein, we have demonstrated that B-cell activation by microbial antigens contributes to the inflammatory and profibrotic milieu seen in patients with IPF. B-cell stimulation by CpG and β-glucan via PRRs resulted in activation of mTOR-dependent and independent pathways. Moreover, we showed that the B-cell–secreted inflammatory milieu is specific to the inducing antigen and causes differential fibroblast migration and activation. B-cell responses to infectious agents and subsequent B-cell–mediated fibroblast activation are modifiable by antifibrotics, but each seems to exert a specific and different effect. These results suggest that, upon PRR activation by microbial antigens, B-cells can contribute to the inflammatory and fibrotic changes seen in patients with IPF, and antifibrotics are able to at least partially reverse these responses.

Abstract B lymphocytes play an essential regulatory role in the adaptive immune response through Ab production during infection. A less known function of B lymphocytes is their ability to respond directly to infectious Ags through stimulation of pattern recognition receptors expressed on their surfaces. β-Glucans are carbohydrates present in the cell wall of many pathogenic fungi that can be detected in the peripheral blood of patients during infection. They have been shown to participate in the innate inflammatory response, as they can directly activate peripheral macrophages and dendritic cells. However, their effect as direct stimulators of B lymphocytes has not been yet fully elucidated. The aim of this study was to examine the molecular mechanisms and cytokine profiles generated following β-glucan stimulation of B lymphocytes, compared with the well-established TLR-9 agonist CpG oligodeoxynucleotide (CpG), and study the participation of β-glucan–stimulated B cells in the innate immune response. In this article, we demonstrate that β-glucan–activated B lymphocytes upregulate proinflammatory cytokines (TNF-α, IL-6, and IL-8). Of interest, β-glucan, unlike CpG, had no effect on B lymphocyte proliferation or IgM production. When compared with CpG (TLR9 agonist), β-glucan–activated cells secreted significantly higher levels of IL-8. Furthermore, IL-8 secretion was partially mediated by Dectin-1 and required SYK, MAPKs, and the transcription factors NF-κB and AP-1. Moreover, we observed that conditioned media from β-glucan–stimulated B lymphocytes elicited neutrophil chemotaxis. These studies suggest that β-glucan–activated B lymphocytes have an important and novel role in fungal innate immune responses.