Use of Clinical Practice Guideline to Improve Management of Osteoarticular Infections in Children.

OBJECTIVES: Osteoarticular infections (OAIs) in children pose significant risks if incorrectly managed. We introduced a clinical practice guideline (CPG) to decrease use of broad-spectrum and intravenous (IV) antibiotics for OAI treatment. The primary aims of our project were to decrease the percent of patients with empirical broad cephalosporin use to 10% and decrease IV antibiotic therapy on discharge to 20% while increasing narrow-spectrum oral antibiotic use to 80% within 24 months. METHODS: We used quality improvement methodology to study patients diagnosed with OAIs. Interventions included multidisciplinary workgroup planning, CPG implementation, education, information technology, and stakeholder feedback. Outcome measures were the percentage of patients prescribed empirical broad-spectrum cephalosporins, percent discharged on IV antibiotics, and percent discharged on narrow-spectrum oral antibiotics. Process measures included percent of patients hospitalized on medicine service and infectious diseases consultation. Balancing measures included rates of adverse drug reactions, disease complications, length of stay, and readmission within 90 days. The impact of the interventions was assessed with run and control charts. RESULTS: A total of 330 patients were included over 96 months. The percentage of patients with empirical broad cephalosporin coverage decreased from 47% to 10%, percent discharged on IV antibiotics decreased from 75% to 11%, and percent discharged on narrow-spectrum oral antibiotics increased from 24% to 84%. Adverse drug reactions decreased from 31% to 10%. Rates of complications, readmissions, and length of stay were unchanged. CONCLUSIONS: Through development and implementation of a CPG for OAI management, we demonstrated decreased use of empirical broad-spectrum antibiotics and improved definitive antibiotic management.

Family Caregiver Acceptability of Assessing Caregiver Adverse Childhood Experiences (ACEs) and Distress in Pediatric Specialty Care.

INTRODUCTION: Providing quality healthcare for children includes assessing and responding to needs of their family caregivers. Three salient domains to consider are caregivers' early adverse childhood experiences (ACEs), their current levels of distress, and their resilience in coping with both prior and current stressors. OBJECTIVE: Determine acceptability of assessing caregiver ACEs, current distress, and resilience in pediatric subspecialty care settings. METHODS: Caregivers of patients in two pediatric specialty care clinics completed questionnaires about their ACEs, recent emotional distress, and resilience. Importantly, caregivers also rated the acceptability of being asked these questions. Participants included 100 caregivers of youth ages 3-17 across Sickle Cell Disease and Pain clinic settings. The majority of participants were mothers (91.0%) who identified as non-Hispanic (86.0%). Caregiver race was primarily African American/Black (53.0%) and White (41.0%). The Area Deprivation Index (ADI) was used to assess socioeconomic disadvantage. RESULTS: High levels of caregiver acceptability or neutrality with assessing ACEs and distress, as well as high ACEs, distress, and resilience. Associations were found between caregiver ratings of acceptability with caregiver resilience and socioeconomic disadvantage. Caregivers reported openness to being asked about their experiences during childhood and recent emotional distress, although ratings of acceptability varied according to other contextual variables, such as level of socioeconomic disadvantage and caregiver resilience. In general, caregivers perceived themselves to be resilient in the face of adversity. CONCLUSIONS: Assessing caregiver ACEs and distress in a trauma-informed way may provide opportunities for better understanding the needs of caregivers and families in order to support them more effectively in the pediatric setting.

Rituximab and antimetabolite treatment of granulomatous and lymphocytic interstitial lung disease in common variable immunodeficiency.

BACKGROUND: Granulomatous and lymphocytic interstitial lung disease (GLILD) is a life-threatening complication in patients with common variable immunodeficiency (CVID), but the optimal treatment is unknown. OBJECTIVE: Our aim was to determine whether rituximab with azathioprine or mycophenolate mofetil improves the high-resolution computed tomography (HRCT) chest scans and/or pulmonary function test results in patients with CVID and GLILD. METHODS: A retrospective chart review of clinical and laboratory data on 39 patients with CVID and GLILD who completed immunosuppressive therapy was performed. Chest HRCT scans, performed before therapy and after the conclusion of therapy, were blinded, randomized, and scored independently by 2 radiologists. Differences between pretreatment and posttreatment HRCT scan scores, pulmonary function test results, and lymphocyte subsets were analyzed. Whole exome sequencing was performed on all patients. RESULTS: Immunosuppressive therapy improved patients' HRCT scan scores (P < .0001), forced vital capacity (P = .0017), FEV1 (P = .037), and total lung capacity (P = .013) but not their lung carbon monoxide diffusion capacity (P = .12). Nine patients relapsed and 6 completed retreatment, with 5 of 6 of these patients (83%) having improved HRCT scan scores (P = .063). Relapse was associated with an increased number of B cells (P = .016) and activated CD4 T cells (P = .016). Four patients (10%) had pneumonia while undergoing active treatment, and 2 patients (5%) died after completion of therapy. Eight patients (21%) had a damaging mutation in a gene known to predispose (TNFRSF13B [n = 3]) or cause a CVID-like primary immunodeficiency (CTLA4 [n = 2], KMT2D [n = 2], or BIRC4 [n = 1]). Immunosuppression improved the HRCT scan scores in patients with (P = .0078) and without (P < .0001) a damaging mutation. CONCLUSIONS: Immunosuppressive therapy improved the radiographic abnormalities and pulmonary function of patients with GLILD. A majority of patients had sustained remissions.

Effect of phosphorylation on EGFR dimer stability probed by single-molecule dynamics and FRET/FLIM.

Deregulation of epidermal growth factor receptor (EGFR) signaling has been correlated with the development of a variety of human carcinomas. EGF-induced receptor dimerization and consequent trans- auto-phosphorylation are among the earliest events in signal transduction. Binding of EGF is thought to induce a conformational change that consequently unfolds an ectodomain loop required for dimerization indirectly. It may also induce important allosteric changes in the cytoplasmic domain. Despite extensive knowledge on the physiological activation of EGFR, the effect of targeted therapies on receptor conformation is not known and this particular aspect of receptor function, which can potentially be influenced by drug treatment, may in part explain the heterogeneous clinical response among cancer patients. Here, we used Förster resonance energy transfer/fluorescence lifetime imaging microscopy (FRET/FLIM) combined with two-color single-molecule tracking to study the effect of ATP-competitive small molecule tyrosine kinase inhibitors (TKIs) and phosphatase-based manipulation of EGFR phosphorylation on live cells. The distribution of dimer on-times was fitted to a monoexponential to extract dimer off-rates (koff). Our data show that pretreatment with gefitinib (active conformation binder) stabilizes the EGFR ligand-bound homodimer. Overexpression of EGFR-specific DEP-1 phosphatase was also found to have a stabilizing effect on the homodimer. No significant difference in the koff of the dimer could be detected when an anti-EGFR antibody (425 Snap single-chain variable fragment) that allows for dimerization of ligand-bound receptors, but not phosphorylation, was used. These results suggest that both the conformation of the extracellular domain and phosphorylation status of the receptor are involved in modulating the stability of the dimer. The relative fractions of these two EGFR subpopulations (interacting versus free) were obtained by a fractional-intensity analysis of ensemble FRET/FLIM images. Our combined imaging approach showed that both the fraction and affinity (surrogate of conformation at a single-molecule level) increased after gefitinib pretreatment or DEP-1 phosphatase overexpression. Using an EGFR mutation (I706Q, V948R) that perturbs the ability of EGFR to dimerize intracellularly, we showed that a modest drug-induced increase in the fraction/stability of the EGFR homodimer may have a significant biological impact on the tumor cell's proliferation potential.

The ErbB4 CYT2 variant protects EGFR from ligand-induced degradation to enhance cancer cell motility.

The epidermal growth factor receptor (EGFR) is a member of the ErbB family that can promote the migration and proliferation of breast cancer cells. Therapies that target EGFR can promote the dimerization of EGFR with other ErbB receptors, which is associated with the development of drug resistance. Understanding how interactions among ErbB receptors alter EGFR biology could provide avenues for improving cancer therapy. We found that EGFR interacted directly with the CYT1 and CYT2 variants of ErbB4 and the membrane-anchored intracellular domain (mICD). The CYT2 variant, but not the CYT1 variant, protected EGFR from ligand-induced degradation by competing with EGFR for binding to a complex containing the E3 ubiquitin ligase c-Cbl and the adaptor Grb2. Cultured breast cancer cells overexpressing both EGFR and ErbB4 CYT2 mICD exhibited increased migration. With molecular modeling, we identified residues involved in stabilizing the EGFR dimer. Mutation of these residues in the dimer interface destabilized the complex in cells and abrogated growth factor-stimulated cell migration. An exon array analysis of 155 breast tumors revealed that the relative mRNA abundance of the ErbB4 CYT2 variant was increased in ER+ HER2- breast cancer patients, suggesting that our findings could be clinically relevant. We propose a mechanism whereby competition for binding to c-Cbl in an ErbB signaling heterodimer promotes migration in response to a growth factor gradient.

Amplification of DNA damage by a γH2AX-targeted radiopharmaceutical.

UNLABELLED: (111)In-DTPA-anti-γH2AX-Tat, which combines an anti-γH2AX antibody with a cell-penetrating peptide, Tat, and the Auger electron-emitting radioisotope, (111)In, targets the DNA damage signalling protein, γH2AX, and has potential as a probe for imaging DNA damage in vivo. The goal of this study was to investigate whether (111)In-DTPA-anti-γH2AX-Tat labelled to high specific activity (6MBq/µg) can amplify treatment-related DNA damage for therapeutic gain. METHODS: MDA-MB-468 and MDA-MB-231/H2N (231-H2N) breast cancer cells were incubated with (111)In-DTPA-anti-γH2AX-Tat (3MBq, 6MBq/µg) or a control radioimmunoconjugate, (111)In-DTPA-mIgG-Tat, and exposed to IR or bleomycin. DNA damage was studied by counting γH2AX foci and by neutral comet assay. Cytotoxicity was evaluated using clonogenic assays. (111)In-DTPA-anti-γH2AX-Tat was administered intravenously to 231-H2N-xenograft-bearing Balb/c nu/nu mice in tumor growth inhibition studies. RESULTS: The number of γH2AX foci was greater after exposure of cells to IR (10Gy) plus (111)In-DTPA-anti-γH2AX-Tat compared to IR alone (20.6±2.5 versus 10.4±2.3 foci/cell; P<.001).(111)In-DTPA-anti-γH2AX-Tat resulted in a reduced surviving fraction in cells co-treated with IR (4Gy) versus IR alone (5.2%±0.9% versus 47.8%±2.8%; P<.001). Similarly, bleomycin (25-200µg/mL) plus (111)In-DTPA-anti-γH2AX-Tat resulted in a lower SF compared to bleomycin alone. The combination of a single exposure to IR (10Gy) plus (111)In-DTPA-anti-γH2AX-Tat significantly decreased the growth rate of 231-H2N xenografts in vivo compared to either (111)In-DTPA-anti-γH2AX-Tat or IR alone (-0.002±0.004 versus 0.036±0.011 and 0.031±0.014mm(3)/day, respectively, P<.001). CONCLUSION: (111)In-DTPA-anti-γH2AX-Tat amplifies anticancer treatment-related DNA damage in vitro and has a potent anti-tumor effect when combined with IR in vivo.

Chemosensitization of cancer cells by KU-0060648, a dual inhibitor of DNA-PK and PI-3K.

DNA double-strand breaks (DSB) are the most cytotoxic lesions induced by topoisomerase II poisons. Nonhomologous end joining (NHEJ) is a major pathway for DSB repair and requires DNA-dependent protein kinase (DNA-PK) activity. DNA-PK catalytic subunit (DNA-PKcs) is structurally similar to PI-3K, which promotes cell survival and proliferation and is upregulated in many cancers. KU-0060648 is a dual inhibitor of DNA-PK and PI-3K in vitro. KU-0060648 was investigated in a panel of human breast and colon cancer cells. The compound inhibited cellular DNA-PK autophosphorylation with IC(50) values of 0.019 µmol/L (MCF7 cells) and 0.17 µmol/L (SW620 cells), and PI-3K-mediated AKT phosphorylation with IC(50) values of 0.039 µmol/L (MCF7 cells) and more than 10 µmol/L (SW620 cells). Five-day exposure to 1 µmol/L KU-0060648 inhibited cell proliferation by more than 95% in MCF7 cells but only by 55% in SW620 cells. In clonogenic survival assays, KU-0060648 increased the cytotoxicity of etoposide and doxorubicin across the panel of DNA-PKcs-proficient cells, but not in DNA-PKcs-deficient cells, thus confirming that enhanced cytotoxicity was due to DNA-PK inhibition. In mice bearing SW620 and MCF7 xenografts, concentrations of KU-0060648 that were sufficient for in vitro growth inhibition and chemosensitization were maintained within the tumor for at least 4 hours at nontoxic doses. KU-0060648 alone delayed the growth of MCF7 xenografts and increased etoposide-induced tumor growth delay in both in SW620 and MCF7 xenografts by up to 4.5-fold, without exacerbating etoposide toxicity to unacceptable levels. The proof-of-principle in vitro and in vivo chemosensitization with KU-0060648 justifies further evaluation of dual DNA-PK and PI-3K inhibitors.

Visualization of a DNA-PK/PARP1 complex.

The DNA-dependent protein kinase (DNA-PK) and Poly(ADP-ribose) polymerase-1 (PARP1) are critical enzymes that reduce genomic damage caused by DNA lesions. They are both activated by DNA strand breaks generated by physiological and environmental factors, and they have been shown to interact. Here, we report in vivo evidence that DNA-PK and PARP1 are equally necessary for rapid repair. We purified a DNA-PK/PARP1 complex loaded on DNA and performed electron microscopy and single particle analysis on its tetrameric and dimer-of-tetramers forms. By comparison with the DNA-PK holoenzyme and fitting crystallographic structures, we see that the PARP1 density is in close contact with the Ku subunit. Crucially, PARP1 binding elicits substantial conformational changes in the DNA-PK synaptic dimer assembly. Taken together, our data support a functional, in-pathway role for DNA-PK and PARP1 in double-strand break (DSB) repair. We also propose a NHEJ model where protein-protein interactions alter substantially the architecture of DNA-PK dimers at DSBs, to trigger subsequent interactions or enzymatic reactions.