Farnesol decreases biofilms of Staphylococcus epidermidis and exhibits synergy with nafcillin and vancomycin.

Biofilm infections are frequently caused by Staphylococcus epidermidis, are resistant to antimicrobial agents, and adversely affect patient outcomes. We evaluated farnesol (FSL), the Candida quorum-sensing molecule, on S. epidermidis biofilms, in vitro and in vivo. We evaluated ED50, ED75, and ED90 (drug concentrations causing 50%, 75%, and 90% inhibition, respectively) of FSL and evaluated synergy with nafcillin and vancomycin. FSL's effects on morphology of S. epidermidis biofilms were analyzed using confocal microscopy and real-time changes using a bioluminescent strain of S. epidermidis, Xen 43. In mice, effects of FSL treatment on s.c. catheter biofilms; cultures of blood, kidney, and catheter and pericatheter tissues; and bioluminescence in strain Xen 43 were evaluated. FSL inhibited biofilms (ED50 ranged from 0.625 to 2.5 mM) and was synergistic with nafcillin and vancomycin at most combination ratios. FSL significantly decreased biovolume, substratum coverage, and mean thickness of S. epidermidis biofilms. In mice, FSL significantly decreased viable colony counts of S. epidermidis from blood, kidney, and catheter and pericatheter tissues and decreased Xen 43 bioluminescence. We confirmed the antibiofilm effects of FSL both in vitro and in vivo, in a bioluminescent strain and its synergy with antibiotics. FSL may be effective against clinical S. epidermidis biofilm infections.

Osteopontin overproduction is associated with progression of glomerular fibrosis in a rat model of anti-glomerular basement membrane glomerulonephritis.

BACKGROUND: Glomerular fibrosis is the common end result of glomerulonephritis (GN) regardless of etiology. In our rat model for anti-glomerular basement membrane GN, severe fibrosis follows glomerular inflammation. We investigated the association between expression of extracellular matrix (ECM) proteins and progression of glomerular fibrosis. METHODS: Expression of ECM genes in glomeruli was determined at RNA and protein levels. Immunofluorescence was applied to identify cell sources for the molecules. RESULTS: DNA microarray for ECM genes, quantitative RT-PCR and Western blot revealed significant upregulation of osteopontin (OPN), a multifunctional molecule, in the glomeruli only after onset of glomerular fibrosis. Two-dimensional electrophoresis showed that the expressed OPN was in three major isoforms. Immunofluorescence showed that fibrotic tissues in glomeruli accumulated massive deposits of extracellular OPN. Both in vivo and in vitro experiments showed that a novel population of multinucleated α-smooth muscle actin(+)CD90(-) myofibroblast-like cells, which surrounded fibrotic tissue, was the main source of OPN during progression of fibrosis. Since senescence-associated ß-galactosidase activity was detected in those cells both in vitro and in vivo, these cells probably were terminally differentiated senescent myofibroblasts. CONCLUSION: OPN has been implicated in fibrosis in several organs. Our results suggest potential roles of OPN and its main source, the senescent myofibroblasts, in glomerular fibrosis.

Blockade of osteopontin inhibits glomerular fibrosis in a model of anti-glomerular basement membrane glomerulonephritis.

BACKGROUND: In our rat model for anti-GBM GN, severe fibrosis follows glomerular inflammation. A potential role of extracellular matrix protein osteopontin (OPN) in glomerular fibrosis was investigated. METHODS: Neutralizing OPN antiserum or control normal serum was injected into the experimental rats at late inflammatory/early fibrotic stage. Glomerular inflammation and fibrosis were determined. RESULTS: OPN antiserum treatment had little effect on glomerular inflammation. However, the antiserum treatment resulted in a significant reduction in number of fibrotic glomeruli (50% of the controls). Histology observation showed that fibrotic tissue in glomeruli of the antiserum treated rats was mild and poorly developed. OPN antiserum treatment resulted in downregulated glomerular expression of collagen 1α1; collagen deposition in the antiserum treated rats reduced to <30% of that for normal serum controls. CONCLUSION: Neutralization of OPN inhibited progression of fibrosis in vivo when given at early fibrotic stage. Thus, OPN may be a therapeutic target for glomerular fibrosis.

Total knee arthroplasty with asymmetric femoral condyles and tibial tray.

Total knee arthroplasties with an asymmetric tibial tray and posterior femoral condyles were implanted in 224 knees. Follow-up averaged 6.3 years. All components were cemented, all patellae were resurfaced, and all femoral components were cruciate-substituting. Postoperative alignment averaged 3.3 degrees valgus. Radiolucencies were absent around 116 knees (66%). No components were radiographically loose. Knee Society scores averaged 85 postoperatively and modified Western Ontario and McMaster Universities Index scores averaged 82. Postoperative flexion averaged 114 degrees. Ten knees required manipulation for arthrofibrosis. Lateral release was necessary in 62 knees (28%). No revisions were required for aseptic loosening. Survivorship free of additional surgical procedures was 97% at 5 years. Compared with other series by designing surgeons, similar excellent survivorship was seen but with a higher lateral release rate.

Mislocalized cytoplasmic p27 activates PAK1-mediated metastasis and is a prognostic factor in osteosarcoma.

The development of pulmonary metastasis is the leading cause of death in osteosarcoma (OS), which is the most common malignant bone tumor in children. We have previously reported that the tumor suppressor p27 (KIP1, CDKN1B) is frequently mislocalized to the cytoplasm of OS. However, its prognostic significance and metastatic mechanism are still elusive. Here, we show that cytoplasmic p27 significantly correlated with a higher metastatic status and poorer survival of OS patients (n = 136, P < 0.05), highlighting the clinical significance of p27 mislocalization in OS. Mechanistically, cytoplasmic p27 is co-immunoprecipitated with p21-activated kinase 1 (PAK1), which resulted in higher PAK1 phosphorylations, actin polymerization, and cell motility in p27-mislocalized OS cells. Silencing PAK1 expression in different p27-mislocalized OS cell lines decreased the migratory and adhesion abilities in vitro, as well as the development of pulmonary metastases in vivo. Similar PAK1-dependent motility was also observed in other p27-mislocalized cancer cell lines. In summary, our study suggests that cytoplasmic p27-mediated PAK1 activation is crucial for OS metastasis. A biomarker-guided targeted therapeutic approach for metastatic OS and other cancers harboring p27 mislocalization can be developed, where cytoplasmic p27 is used for risk stratification and PAK1 can be exploited as a potential therapeutic target.

Engineering human tumor-specific cytotoxic T cells to function in a hypoxic environment.

Hypoxia occurs in many tumors and reduces the effectiveness of radio- and chemotherapy. Hypoxia also impedes immune responses to tumors, reducing T lymphocyte production of cytokines such as interleukin-2 (IL-2) and interferon gamma, as well as the survival and proliferation of these cells. We constructed a lentiviral vector encoding a bidirectional hypoxia-inducible responsive element (HRE) derived from human vascular endothelial growth factor, which drives the hIL-2 gene and a marker gene. We used a model of human B cell lymphoma to show that tumor-specific T cells modified with this vector upregulate hIL-2 expression when oxygen tension is low in vitro and in vivo. The consequence of this effect is to increase T-cell survival and proliferation whilst sustaining effector function, even in O(2) concentrations as low as 1%. The phenotype of the transduced cells is unchanged, as is their ability to migrate to tumor. HRE-IL-2-modified cytotoxic T lymphocytes (CTLs) produce faster and more complete tumor regression than parental CTLs and increase overall survival. Hypoxia-resistant T cells may thus be of value in the treatment of human tumors in which areas of hypoxia may otherwise account for resistance to this therapeutic strategy.

Activating MAPK1 (ERK2) mutation in an aggressive case of disseminated juvenile xanthogranuloma.

Juvenile xanthogranuloma (JXG) is a rare histiocytic disorder that is usually benign and self-limiting. We present a case of atypical, aggressive JXG harboring a novel mitogen-activated protein kinase (MAPK) pathway mutation in the MAPK1 gene, which encodes mitogen-activated protein kinase 1 or extracellular signal-regulated 2 (ERK2). Our analysis revealed that the mutation results in constitutive ERK activation that is resistant to BRAF or MEK inhibitors but susceptible to an ERK inhibitor. These data highlight the importance of identifying specific MAPK pathway alterations as part of the diagnostic workup for patients with histiocytic disorders rather than initiating empiric treatment with MEK inhibitors.

p27 Is a Candidate Prognostic Biomarker and Metastatic Promoter in Osteosarcoma.

Metastatic progression is the major cause of death in osteosarcoma, the most common bone malignancy in children and young adults. However, prognostic biomarkers and efficacious targeted treatments for metastatic disease remain lacking. Using an immunoproteomic approach, we discovered that autoantibodies against the cell-cycle kinase inhibitor p27 (KIP1, CDKN1B) were elevated in plasma of high-risk osteosarcoma patients. Using a large cohort of serum samples from osteosarcoma patients (n = 233), we validated that a higher level of the p27 autoantibody significantly correlated with poor overall and event-free survival (P < 0.05). Immunohistochemical analysis also showed that p27 was mislocalized to the cytoplasm in the majority of osteosarcoma cases and in highly metastatic osteosarcoma cell lines. We demonstrated that ectopic expression of cytoplasmic p27 promoted migration and invasion of osteosarcoma cells, whereas shRNA-mediated gene silencing suppressed these effects. In addition, mutations at the p27 phosphorylation sites S10 or T198, but not T157, abolished the migratory and invasive phenotypes. Furthermore, the development of pulmonary metastases increased in mice injected with cells expressing cytoplasmic p27 compared with an empty vector control. Collectively, our findings support further investigation of p27 as a potential prognostic biomarker and therapeutic target in osteosarcoma cases exhibiting aberrant p27 subcellular localization. Cancer Res; 76(13); 4002-11. ©2016 AACR.

Prognostic Significance of Major Histocompatibility Complex Class II Expression in Pediatric Adrenocortical Tumors: A St. Jude and Children's Oncology Group Study.

PURPOSE: Histologic markers that differentiate benign and malignant pediatric adrenocortical tumors are lacking. Previous studies have implicated an association of MHC class II expression with adrenocortical tumor prognosis. Here, we determined the expression of MHC class II as well as the cell of origin of these immunologic markers in pediatric adrenocortical tumor. The impact of MHC class II gene expression on outcome was determined in a cohort of uniformly treated children with adrenocortical carcinomas. EXPERIMENTAL DESIGN: We analyzed the expression of MHC class II and a selected cluster of differentiation genes in 63 pediatric adrenocortical tumors by Affymetrix Human U133 Plus 2.0 or HT HG-U133+PM gene chip analyses. Cells expressing MHC class II were identified by morphologic and immunohistochemical assays. RESULTS: MHC class II expression was significantly greater in adrenocortical adenomas than in carcinomas (P = 4.8 ×10-6) and was associated with a higher progression-free survival (PFS) estimate (P = 0.003). Specifically, HLA-DPA1 expression was most significantly associated with PFS after adjustment for tumor weight and stage. HLA-DPA1 was predominantly expressed by hematopoietic infiltrating cells and undetectable in tumor cells in 23 of 26 cases (88%). CONCLUSIONS: MHC class II expression, which is produced by tumor-infiltrating immune cells, is an indicator of disease aggressiveness in pediatric adrenocortical tumor. Our results suggest that immune responses modulate adrenocortical tumorigenesis and may allow the refinement of risk stratification and treatment for this disease. Clin Cancer Res; 22(24); 6247-55. ©2016 AACR.

Cancer-Associated Fibroblasts Induce a Collagen Cross-link Switch in Tumor Stroma.

UNLABELLED: Intratumoral collagen cross-links heighten stromal stiffness and stimulate tumor cell invasion, but it is unclear how collagen cross-linking is regulated in epithelial tumors. To address this question, we used Kras(LA1) mice, which develop lung adenocarcinomas from somatic activation of a Kras(G12D) allele. The lung tumors in Kras(LA1) mice were highly fibrotic and contained cancer-associated fibroblasts (CAF) that produced collagen and generated stiffness in collagen gels. In xenograft tumors generated by injection of wild-type mice with lung adenocarcinoma cells alone or in combination with CAFs, the total concentration of collagen cross-links was the same in tumors generated with or without CAFs, but coinjected tumors had higher hydroxylysine aldehyde-derived collagen cross-links (HLCC) and lower lysine-aldehyde-derived collagen cross-links (LCCs). Therefore, we postulated that an LCC-to-HLCC switch induced by CAFs promotes the migratory and invasive properties of lung adenocarcinoma cells. To test this hypothesis, we created coculture models in which CAFs are positioned interstitially or peripherally in tumor cell aggregates, mimicking distinct spatial orientations of CAFs in human lung cancer. In both contexts, CAFs enhanced the invasive properties of tumor cells in three-dimensional (3D) collagen gels. Tumor cell aggregates that attached to CAF networks on a Matrigel surface dissociated and migrated on the networks. Lysyl hydroxylase 2 (PLOD2/LH2), which drives HLCC formation, was expressed in CAFs, and LH2 depletion abrogated the ability of CAFs to promote tumor cell invasion and migration. IMPLICATIONS: CAFs induce a collagen cross-link switch in tumor stroma to influence the invasive properties of tumor cells.

An overview of terrorism and its impact on biomedical research facilities.

Since the '9/11' and anthrax-contaminated-letter events of 2001, American society has given the term "bioterrorism" much attention. The author clarifies the definitions associated with bioterrorism and terrorism, provides an historical perspective regarding bioweapons, defines and characterizes the types of agents used as bioweapons, reviews pertinent bioterrorism legislation, and concludes by assessing the impact of these elements on biomedical research facilities.

Fibulin-2 is a driver of malignant progression in lung adenocarcinoma.

The extracellular matrix of epithelial tumors undergoes structural remodeling during periods of uncontrolled growth, creating regional heterogeneity and torsional stress. How matrix integrity is maintained in the face of dynamic biophysical forces is largely undefined. Here we investigated the role of fibulin-2, a matrix glycoprotein that functions biomechanically as an inter-molecular clasp and thereby facilitates supra-molecular assembly. Fibulin-2 was abundant in the extracellular matrix of human lung adenocarcinomas and was highly expressed in tumor cell lines derived from mice that develop metastatic lung adenocarcinoma from co-expression of mutant K-ras and p53. Loss-of-function experiments in tumor cells revealed that fibulin-2 was required for tumor cells to grow and metastasize in syngeneic mice, a surprising finding given that other intra-tumoral cell types are known to secrete fibulin-2. However, tumor cells grew and metastasized equally well in Fbln2-null and -wild-type littermates, implying that malignant progression was dependent specifically upon tumor cell-derived fibulin-2, which could not be offset by other cellular sources of fibulin-2. Fibulin-2 deficiency impaired the ability of tumor cells to migrate and invade in Boyden chambers, to create a stiff extracellular matrix in mice, to cross-link secreted collagen, and to adhere to collagen. We conclude that fibulin-2 is a driver of malignant progression in lung adenocarcinoma and plays an unexpected role in collagen cross-linking and tumor cell adherence to collagen.

Thrombotic microangiopathic hemolytic anemia with reduction of ADAMTS13 activity: initial manifestation of childhood-onset systemic lupus erythematosus.

Severe manifestations of systemic lupus erythematosus (SLE), antiphospholipid syndrome (APS), and thrombotic thrombocytopenic purpura (TTP) are characterized by multiorgan thrombotic microangiopathy. We describe reduction of ADAMTS13 activity and the development of systemic autoimmunity in all 8 children initially diagnosed with acquired noncongenital TTP during an 8.5-year period. Median age at diagnosis was 12.0 years (range, 2.6-17.3 years). ADAMTS13 activity was absent (<5%) in 6 patients; 3 patients had a detected inhibitor. SLE was diagnosed concurrently in 3 patients, and 4 patients were diagnosed within 5 years. Six of the children diagnosed with SLE had absent ADAMTS13 activity at diagnosis. In 6 patients with SLE, immune-mediated nephritis developed by 46 months. All surviving patients with SLE developed antiphospholipid antibodies, including some with a lupus anticoagulant. Patients with SLE did not have TTP recurrences once daily immunosuppressive regimens were started. An evaluation for SLE/APS is warranted in children and adolescents with reduced ADAMTS13 activity and thrombotic microangiopathy.

Complications of pedicle screw fixation in scoliosis surgery: a systematic review.

STUDY DESIGN: Systematic review. OBJECTIVE: To review the published literature on the use of pedicle screws in pediatric spinal deformity to quantify the risks and complications associated with pedicle screw instrumentation, particularly in the thoracic spine. SUMMARY OF BACKGROUND DATA: The use of pedicle screws in adolescent scoliosis surgery is common. Although many reports have been published regarding the use of pedicle screws in pediatric patients, there has been no systematic review on the risks of complications. METHODS: PubMed, Ovid Medline, and Cochrane databases were searched for studies reporting the use of thoracic pedicle screws in pediatric deformity. We excluded articles dealing with neuromuscular scoliosis or bone dysplasia to focus mostly on adolescent thoracic idiopathic scoliosis and the likes. We then searched for cases reports dealing with thoracic pedicle screws complications. RESULTS: This systematic review retrieved 21 studies with a total of 4570 pedicle screws in 1666 patients. The mean age of the patients was 17.6 years; 812 patients were women and 252 were men, and 5 studies did not identify sex. Overall, 518 (4.2%) screws were reported as malpositioned. However, in studies in which postoperative computed tomography scans were done systematically, the rate of screw malpositioning was as high as 15.7%. The reported percentage of patients with screw malpositioned is around 11%. Eleven patients underwent revision surgery for instrumentation malposition. Other complications reported include loss of curve correction, intraoperative pedicle fracture or loosening, dural laceration, deep infection, pseudarthrosis, and transient neurologic injury. There were no major vascular complications reported in these 21 studies. We could identify 9 case report articles dealing with complications of pedicle screws. Such complications were mostly either vascular (10 cases) or neurologic (4 cases), without any irreversible complications. CONCLUSION: Malposition is the most commonly reported complication of thoracic pedicle screw placement, at a rate of 15.7% per screw inserted with postoperative computed tomography scans. The use of pedicle screws in the thoracic spine for the treatment of pediatric deformity has been reported to be safe despite the high rate of patients with malpositioned screws (11%). Major complications, such as neurologic or vascular injury, were almost never reported in this literature review of case series. Cases reports on the other hand have started to identify such complications.

Optimal utilization of histopathologic analysis of tonsil and adenoid specimens in the pediatric population.

OBJECTIVE: To evaluate the optimal utilization of histopathologic analysis of tonsil and adenoid specimens in the pediatric population. METHODS: A retrospective review was performed on 7837 tonsil and adenoid specimens submitted from January 2004 to April 2008. The records were reviewed for the patients' age, sex, and pathologic analysis. The time and cost per analysis of each specimen were determined. RESULTS: Histopathology was performed on 347 specimens based on clinical suspicion by the surgeon, a difference of 0.5 cm or more among tonsils, gross abnormalities, and history of malignancy, transplant, or immunocompromise. Malignancy was diagnosed in 0.026% of patients. Post-transplant lymphoproliferative disease was diagnosed in 6 of 24 immunocompromised patients. The use of these criteria resulted in a savings of $518,088.47 and 461 h of dedicated technician time per year. CONCLUSIONS: Histologic examinations in selected specimens should be based on specific criteria that should be determined by each hospital based on hospital size, finances and input from their pathologists and otolaryngogists. Storage of a representative specimen for possible retrospective review may be useful.

The reliability of the "Scratch test".

The "Scratch Test" uses a sharp scalpel to scrape areas of suspected tendinosis in the management of lateral and medial epicondylitis. As claimed in the literature, this tissue is friable and peels off, whereas normal tendon does not. The purpose of this study was to determine whether, or not, the "Scratch Test" is able to differentiate between tendinosis and more normal adjacent tendon. Nineteen specimens from patients treated for tendinosis about the elbow were examined histologically. Three groups of specimens were compared: (1) grossly abnormal tendon, (2) tendon that was scraped out using the "Scratch Test" and (3) tendon that remained behind following the "Scratch Test". There was no significant histological difference between visibly degenerated tendon (group 1) and that which was scraped out using the "Scratch Test" (group 2). There was, however, a significant histological difference between both these groups and the more normal tendon tissue that the "Scratch Test" left behind (group 3).