Development of an automated amplicon-based next-generation sequencing pipeline for rapid detection of bacteria and fungi directly from clinical specimens.

Unlabelled: The timely identification of microbial pathogens is essential to guide targeted antimicrobial therapy and ultimately, successful treatment of an infection. However, the yield of standard microbiology testing (SMT) is directly related to the duration of antecedent antimicrobial therapy as SMT culture methods are dependent on the recovery of viable organisms, the fastidious nature of certain pathogens, and other pre-analytic factors. In the last decade, metagenomic next-generation sequencing (mNGS) has been successfully utilized as a diagnostic tool for various applications within the clinical laboratory.

Clinical utility of SARS-CoV-2 subgenomic RT-PCR in a pediatric quaternary care setting.

Background: During active transcription, SARS-CoV-2 generates subgenomic regions of viral RNA. While standard SARS-CoV-2 RT-PCR amplifies region(s) of genomic RNA, it cannot distinguish active infection from remnant viral genomic material. However, screening for subgenomic RNA (sgRNA) by RT-PCR may aid in the determination of actively transcribing virus.

Molecular Testing for Detection of Groups A, C, and G β-Hemolytic Streptococci in Pharyngeal Samples from Children.

Background: Group A Streptococcus (GAS) and large colony-forming group C (GCS) and G (GGS) β-hemolytic streptococci are important causes of acute pharyngitis in children and adults. Rapid and accurate diagnosis of streptococcal pharyngitis can improve patient care and potentially reduce transmission. In this study, we evaluated the performance of the Lyra Direct Strep (LDS) assay for detection of GAS and GCS/GGS compared with traditional culture methods.

A 5-year study of the performance of the Verigene Gram-positive blood culture panel in a pediatric hospital.

High accuracy of direct from positive blood culture molecular panels is imperative, particularly for the detection of resistance determinants as it allows for antimicrobial optimization prior to conventional susceptibility testing. In this study, we provide extensive data since implementation of the Verigene Gram-positive blood culture panel (BC-GP) in 2013. Within 5 years, 1636 blood culture bottles positive for a Gram-positive organism were tested on the BC-GP panel.

One Year in the Life of a Rapid Syndromic Panel for Meningitis/Encephalitis: a Pediatric Tertiary Care Facility's Experience.

Early establishment of infectious processes allows for expedited clinical management of meningitis and encephalitis. The FilmArray meningitis/encephalitis (FA-M/E) panel provides rapid detection of potential pathogens associated with encephalitis/meningitis in both immunocompetent and compromised patients. Here, we conducted a 1-year review of the performance of the FA-M/E panel at a tertiary care children's hospital.

Multicenter Evaluation of BioFire FilmArray Meningitis/Encephalitis Panel for Detection of Bacteria, Viruses, and Yeast in Cerebrospinal Fluid Specimens.

Rapid diagnosis and treatment of infectious meningitis and encephalitis are critical to minimize morbidity and mortality. Comprehensive testing of cerebrospinal fluid (CSF) often includes Gram stain, culture, antigen detection, and molecular methods, paired with chemical and cellular analyses. These methods may lack sensitivity or specificity, can take several days, and require significant volume for complete analysis.

Direct Identification of Aerobic Bacteria by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry Is Accurate and Robust.

Background: Bacterial identification in the clinical laboratory can be laborious and expensive. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a rapid and cost-effective diagnostic tool for the identification of organisms routinely found in the microbiology laboratory. The objective of this study was to demonstrate that identification of aerobic Gram-positive and Gram-negative organisms could be performed accurately and efficiently by MALDI-TOF MS and the Bruker Biotyper system without the use of time-consuming extraction methodologies.

Direct identification of bacteria from positive BacT/ALERT blood culture bottles using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is a fast and robust method for the identification of bacteria. In this study, we evaluate the performance of a laboratory-developed lysis method (LDT) for the rapid identification of bacteria from positive BacT/ALERT blood culture bottles. Of the 168 positive bottles tested, 159 were monomicrobial, the majority of which were Gram-positive organisms (61.

Performance of the Verigene Gram-positive blood culture assay for direct detection of Gram-positive organisms and resistance markers in a pediatric hospital.

The performance characteristics of the Verigene Gram-positive blood culture (BC-GP) assay were evaluated in pediatric patients. Concordance of the BC-GP assay was 95.8%, with significant decreases in turnaround time for identification and resistance detection.

Mycophenolate mofetil decreases atherosclerotic lesion size by depression of aortic T-lymphocyte and interleukin-17-mediated macrophage accumulation.

Objectives: This study tested whether immunosuppression with mycophenolate mofetil (MMF) inhibits atherosclerosis development in apolipoprotein-E-deficient (Apoe(-/-)) mice and investigated the mechanism.

Background: Chronic vascular inflammation involving both innate and adaptive immunity is central in the development of atherosclerosis, but immunosuppressive treatment is not uniformly beneficial. The immunosuppressive MMF targets lymphocyte proliferation by inhibiting inosine-monophosphate dehydrogenase.

Monocyte-endothelial cell interactions in the development of atherosclerosis.

The activation of endothelial cells at atherosclerotic lesion-prone sites in the arterial tree results in the up-regulation of cell adhesion molecules and chemokines, which mediate the recruitment of circulating monocytes. Accumulation of monocytes and monocyte-derived phagocytes in the wall of large arteries leads to chronic inflammation and the development and progression of atherosclerosis. This review discusses the nature of these molecules and the mechanisms involved in the early steps of monocyte recruitment into atherosclerotic lesion sites within the vessel wall.

Chapter 11. Intravital microscopic investigation of leukocyte interactions with the blood vessel wall.

Intravital microscopy is a method to study the microcirculation in living tissues. Transillumination, oblique reflected light illumination, continuous and stroboscopic epifluorescence microscopy can be used to visualized specific cells and molecules. Intravital microscopy is further enhanced by the advent of laser scanning.

Stromal derived factor-1 (SDF-1/CXCL12) and CXCR4 in renal cell carcinoma metastasis.

Renal cell carcinoma (RCC) is characterized by organ-specific metastases. The chemokine stromal derived factor-1 (SDF-1/CXCL12) and its receptor CXCR4 have been suggested to regulate organ-specific metastasis in various other cancers. On this basis, we hypothesized that the biological axis of CXCL12 via interaction with its receptor, CXCR4, is a major mechanism for RCC metastasis.

Cancer CXC chemokine networks and tumour angiogenesis.

Chemokines have pleiotropic effects in regulating immunity, angiogenesis, stem cell trafficking, and mediating organ-specific metastases of cancer. In the context of angiogenesis, the CXC chemokine family is a unique group of cytokines known for their ability to behave in a disparate manner in the regulation of angiogenesis. The glutamic acid-leucine-arginine (ELR+) CXC chemokines are potent promoters of angiogenesis, and mediate their angiogenic activity via signal-coupling of CXCR2 on endothelium.

CXCR2/CXCR2 ligand biology during lung transplant ischemia-reperfusion injury.

Lung transplantation is a therapeutic option for a number of end-stage pulmonary disorders. Early lung allograft dysfunction (ischemia-reperfusion injury) continues to be the most common cause of early mortality after lung transplantation and a significant risk factor for the development of bronchiolitis obliterans syndrome. Ischemia-reperfusion injury is characterized histopathologically by lung edema and a neutrophil predominate leukocyte extravasation.

The role of CXCR2/CXCR2 ligand biological axis in renal cell carcinoma.

Renal cell carcinoma (RCC) accounts for 3% of new cancer incidence and mortality in the United States. Studies in RCC have predominantly focused on VEGF in promoting tumor-associated angiogenesis. However, other angiogenic factors may contribute to the overall angiogenic milieu of RCC.

Endothelial cell co-stimulation through OX40 augments and prolongs T cell cytokine synthesis by stabilization of cytokine mRNA.

Human endothelial cells (ECs) constitutively express OX40L and co-stimulate memory CD4(+) T cell proliferation that is dependent upon OX40-OX40L interaction. In vivo, OX40 prolongs T cell survival; however, an unanswered question is whether it can also prolong synthesis of proliferation-sustaining cytokines such as IL-2. Here we show that EC co-stimulation results in the secretion of T cell IL-2, IL-3 and IFN-gamma and that in the absence of OX40 signals synthesis largely ceases by 12-18 h, but is prolonged up to 60 h in the presence of OX40 signaling.

Role of CXCR2/CXCR2 ligands in vascular remodeling during bronchiolitis obliterans syndrome.

Angiogenesis and vascular remodeling support fibroproliferative processes; however, no study has addressed the importance of angiogenesis during fibro-obliteration of the allograft airway during bronchiolitis obliterans syndrome (BOS) that occurs after lung transplantation. The ELR(+) CXC chemokines both mediate neutrophil recruitment and promote angiogenesis. Their shared endothelial cell receptor is the G-coupled protein receptor CXC chemokine receptor 2 (CXCR2).

Epidermal growth factor and hypoxia-induced expression of CXC chemokine receptor 4 on non-small cell lung cancer cells is regulated by the phosphatidylinositol 3-kinase/PTEN/AKT/mammalian target of rapamycin signaling pathway and activation of hypoxia inducible factor-1alpha.

Non-small cell lung cancer (NSCLC) expresses a particularly aggressive metastatic phenotype, and patients with this disease have a poor prognosis. CXC chemokine receptor 4 (CXCR4) is a cell surface receptor that has been shown to mediate the metastasis of many solid tumors including lung, breast, kidney, and prostate. In addition, overexpression of the epidermal growth factor receptor (EGFR) is associated with the majority of NSCLC and has been implicated in the process of malignant transformation by promoting cell proliferation, cell survival, and motility.

Of mice and not men: differences between mouse and human immunology.

Mice are the experimental tool of choice for the majority of immunologists and the study of their immune responses has yielded tremendous insight into the workings of the human immune system. However, as 65 million years of evolution might suggest, there are significant differences. Here we outline known discrepancies in both innate and adaptive immunity, including: balance of leukocyte subsets, defensins, Toll receptors, inducible NO synthase, the NK inhibitory receptor families Ly49 and KIR, FcR, Ig subsets, the B cell (BLNK, Btk, and lambda5) and T cell (ZAP70 and common gamma-chain) signaling pathway components, Thy-1, gammadelta T cells, cytokines and cytokine receptors, Th1/Th2 differentiation, costimulatory molecule expression and function, Ag-presenting function of endothelial cells, and chemokine and chemokine receptor expression.

Identification of endothelial cell genes expressed in an in vitro model of angiogenesis: induction of ESM-1, (beta)ig-h3, and NrCAM.

Blood vessel growth by angiogenesis plays an essential role in embryonic development, wound healing, and tumor growth. To understand the molecular cues underlying this process we have used the PCR-based subtractive hybridization method, representational difference analysis, to identify genes upregulated in endothelial cells (EC) forming tubes in 3D collagen gels, compared to migrating and proliferating cells in 2D cultures. We identified several previously characterized angiogenic markers, including the alpha(v) chain of the alpha(v)beta3 integrin and plasminogen activator inhibitor-1, suggesting overlap in gene expression between tube-forming cells in vitro and in vivo.