Human Dectin-1 Deficiency Impairs Macrophage-Mediated Defense Against Phaeohyphomycosis

Subcutaneous phaeohyphomycosis typically affects immunocompetent individuals following traumatic inoculation. Severe or disseminated infection can occur in CARD9 deficiency or after transplantation, but the mechanisms protecting against phaeohyphomycosis remain unclear. We evaluated a patient with progressive, refractory Corynespora cassiicola phaeohyphomycosis and found that he carried biallelic deleterious mutations in CLEC7A encoding the CARD9-coupled, β-glucan-binding receptor, Dectin-1. The patient\u27s PBMCs failed to produce TNF-α and IL-1β in response to β-glucan and/or C. cassiicola. To confirm the cellular and molecular requirements for immunity against C. cassiicola, we developed a mouse model of this infection. Mouse macrophages required Dectin-1 and CARD9 for IL-1β and TNF-α production, which enhanced fungal killing in an interdependent manner. Deficiency of either Dectin-1 or CARD9 was associated with more severe fungal disease, recapitulating the human observation. Because these data implicated impaired Dectin-1 responses in susceptibility to phaeohyphomycosis, we evaluated 17 additional unrelated patients with severe forms of the infection. We found that 12 out of 17 carried deleterious CLEC7A mutations associated with an altered Dectin-1 extracellular C-terminal domain and impaired Dectin-1-dependent cytokine production. Thus, we show that Dectin-1 and CARD9 promote protective TNF-α- and IL-1β-mediated macrophage defense against C. cassiicola. More broadly, we demonstrate that human Dectin-1 deficiency may contribute to susceptibility to severe phaeohyphomycosis by certain dematiaceous fungi

The Role of Integrin Alpha9Beta1 During Angiogenesis Induced by VEGF-A

Integrins are heterodimeric membrane proteins that mediate cell adhesion, migration and proliferation in a number of physiologic processes, including angiogenesis. The integrin α9β1 binds to a number of ligands, including the angiogenic growth factor, VEGF-A, and mediates cell adhesion and proliferation on VEGF-A. Blockade of α9β1 inhibits angiogenesis in the chick and quail chorioallantoic membranes, but the specific cell type(s) in which α9β1 is expressed and the mechanisms by which integrin α9β1 mediates angiogenesis have not been described. The complete knockout of α9 results in lethal chylothoraces between postnatal day 6 and 10, but has no reported deficits of vasculogenesis or developmental angiogenesis. To study the role of α9β1 during pathologic angiogenesis, we generated a mouse in which α9 can be temporally deleted in all tissues following administration of Doxycyline. Administration of Doxycycline after the development of the lymphatics allowed us to define a role for α9β1 during pathologic angiogenesis in adult mice. Deletion of α9β1 inhibited angiogenesis in vivo induced by VEGF-A, but not by bFGF. We determined that α9β1 is expressed on cells which also express the pericyte markers NG2 and PDGFRβ, and not on endothelial cells expressing PECAM. Pericyte-specific deletion of the integrin using an α-Smooth Muscle Actin promoter was sufficient to inhibit angiogenesis in response to VEGF-A due to a failure of pericyte recruitment before endothelial cell recruitment. Taken together, these results indicate that pathologic angiogenesis in response to VEGF-A requires pericyte recruitment before endothelial cell recruitment, and that this pericyte recruitment requires expression of integrin α9β1 on pericytes

Mfge8 Is Critical for Mammary Gland Remodeling during Involution

Apoptosis is a critical process in normal mammary gland development and the rapid clearance of apoptotic cells prevents tissue injury associated with the release of intracellular antigens from dying cells. Milk fat globule-EGF-factor 8 (Mfge8) is a milk glycoprotein that is abundantly expressed in the mammary gland epithelium and has been shown to facilitate the clearance of apoptotic lymphocytes by splenic macrophages. We report that mice with disruption of Mfge8 had normal mammary gland development until involution. However, abnormal mammary gland remodeling was observed postlactation in Mfge8 mutant mice. During early involution, Mfge8 mutant mice had increased numbers of apoptotic cells within the mammary gland associated with a delay in alveolar collapse and fat cell repopulation. As involution progressed, Mfge8 mutants developed inflammation as assessed by CD45 and CD11b staining of mammary gland tissue sections. With additional pregnancies, Mfge8 mutant mice developed progressive dilatation of the mammary gland ductal network. These data demonstrate that Mfge8 regulates the clearance of apoptotic epithelial cells during mammary gland involution and that the absence of Mfge8 leads to inflammation and abnormal mammary gland remodeling

A simple solid media assay for detection of synergy between bacteriophages and antibiotics.

The emergence of antibiotic-resistant bacteria (ARB) has necessitated the development of alternative therapies to deal with this global threat. Bacteriophages (viruses that target bacteria) that kill ARB are one such alternative. Although phages have been used clinically for decades with inconsistent results, a number of recent advances in phage selection, propagation, and purification have enabled a reevaluation of their utility in contemporary clinical medicine. In most phage therapy cases, phages are administered in combination with antibiotics to ensure that patients receive the standard-of-care treatment. Some phages may work cooperatively with antibiotics to eradicate ARB, as often determined using non-standardized broth assays. We sought to develop a solid media-based assay to assess cooperativity between antibiotics and phages to offer a standardized platform for such testing. We modeled the interactions that occur between antibiotics and phages on solid medium to measure additive, antagonistic, and synergistic interactions. We then tested the method using different bacterial isolates and identified a number of isolates where synergistic interactions were identified. These interactions were not dependent on the specific organism, phage family, or antibiotic used. A priori susceptibility to the antibiotic or the specific phage were not requirements to observe synergistic interactions. Our data also confirm the potential for the restoration of vancomycin to treat vancomycin-resistant Enterococcus (VRE) when used in combination with phages. Solid media assays for the detection of cooperative interactions between antibiotics and phages can be an accessible technique adopted by clinical laboratories to evaluate antibiotic and phage choices in phage therapy.IMPORTANCEBacteriophages have become an important alternative treatment for individuals with life-threatening antibiotic-resistant bacteria (ARB) infections. Because antibiotics represent the standard-of-care for treatment of ARB, antibiotics and phages often are delivered together without evidence that they work cooperatively. Testing for cooperativity can be difficult due to the equipment necessary and a lack of standardized means for performing the testing in liquid medium. We developed an assay using solid medium to identify interactions between antibiotics and phages for gram-positive and gram-negative bacteria. We modeled the interactions between antibiotics and phages on solid medium, and then tested multiple replicates of vancomycin-resistant Enterococcus (VRE) and Stenotrophomonas in the assay. For each organism, we identified synergy between different phage and antibiotic combinations. The development of this solid media assay for assessing synergy between phages and antibiotics will better inform the use of these combinations in the treatment of ARB infections

Epidemiologic, clinical, and serum markers may improve discrimination between bacterial and viral etiologies of childhood pneumonia

BackgroundDiscrimination of bacterial and viral etiologies of childhood community-acquired pneumonia (CAP) is often challenging. Unnecessary antibiotic administration exposes patients to undue risks and may engender antimicrobial resistance. This study aimed to develop a prediction model using epidemiological, clinical and laboratory data to differentiate between bacterial and viral CAP.MethodsData from 155 children with confirmed bacterial or mixed bacterial and viral infection (N = 124) and viral infection (N = 31) were derived from a comprehensive assessment of causative pathogens [Partnerships for Enhanced Engagement in Research-Pneumonia in Pediatrics (PEER-PePPeS)] conducted in Indonesia. Epidemiologic, clinical and biomarker profiles (hematology and inflammatory markers) were compared between groups. The area under the receiver operating characteristic curve (AUROC) for varying biomarker levels was used to characterize performance and determine cut-off values for discrimination of bacterial and mixed CAP versus viral CAP. Diagnostic predictors of bacterial and mixed CAP were assessed by multivariate logistic regression.ResultsDiarrhea was more frequently reported in bacterial and mixed CAP, while viral infections more frequently occurred during Indonesia’s rainy season. White blood cell counts (WBC), absolute neutrophil counts (ANC), neutrophil-lymphocyte ratio (NLR), C-reactive protein (CRP), and procalcitonin (PCT) were significantly higher in bacterial and mixed cases. After adjusting for covariates, the following were the most important predictors of bacterial or mixed CAP: rainy season (aOR 0.26; 95% CI 0.08–0.90; p = 0.033), CRP ≥5.70 mg/L (aOR 4.71; 95% CI 1.18–18.74; p = 0.028), and presence of fever (aOR 5.26; 95% CI 1.07–25.91; p = 0.041). The model assessed had a low R-squared (Nagelkerke R2 = 0.490) but good calibration (p = 0.610 for Hosmer Lemeshow test). The combination of CRP and fever had moderate predictive value with sensitivity and specificity of 62.28 and 65.52%, respectively.ConclusionCombining clinical and laboratory profiles is potentially valuable for discriminating bacterial and mixed from viral pediatric CAP and may guide antibiotic use. Further studies with a larger sample size should be performed to validate this model