Polymeric nanofiber coating with tunable combinatorial antibiotic delivery prevents biofilm-associated infection in vivo.

Bacterial biofilm formation is a major complication of implantable medical devices that results in therapeutically challenging chronic infections, especially in cases involving antibiotic-resistant bacteria. As an approach to prevent these infections, an electrospun composite coating comprised of poly(lactic-coglycolic acid) (PLGA) nanofibers embedded in a poly(ε-caprolactone) (PCL) film was developed to locally codeliver combinatorial antibiotics from the implant surface. The release of each antibiotic could be adjusted by loading each drug into the different polymers or by varying PLGA:PCL polymer ratios. In a mouse model of biofilm-associated orthopedic-implant infection, three different combinations of antibiotic-loaded coatings were highly effective in preventing infection of the bone/joint tissue and implant biofilm formation and were biocompatible with enhanced osseointegration. This nanofiber composite-coating technology could be used to tailor the delivery of combinatorial antimicrobial agents from various metallic implantable devices or prostheses to effectively decrease biofilm-associated infections in patients.

Apolipoprotein E inhibits osteoclast differentiation via regulation of c-Fos, NFATc1 and NF-κB.

Apolipoprotein E (ApoE) plays a major role in the transport and metabolism of lipid. Other functions of ApoE include modulation of innate and adaptive immune responses. The expression of ApoE in osteoblasts and its relevance with bone formation have also been reported. However, the effect of ApoE on osteoclasts has not yet been examined. Here, we investigated the role of ApoE in osteoclast differentiation using bone marrow-derived macrophages (BMMs) and RAW264.7 cells. We found a down-regulation of ApoE gene expression during osteoclastic differentiation of those cells. Overexpression of ApoE in BMMs and RAW264.7 cells significantly blocked the induction of c-Fos and nuclear factor of activated T cell c1 (NFATc1), transcription factors critical for expression of osteoclast marker genes, by receptor activator of nuclear factor κB ligand (RANKL), the osteoclast differentiation factor. ApoE inhibited osteoclast differentiation, as measured by decreased number of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNCs). In addition, ApoE reduced the expression of dendritic cell-specific transmembrane protein (DC-STAMP) and ATPase, H(+) transporting, lysosomal 38kDa, V0 subunit d2 (ATP6v0d2), genes involved in cell-cell fusion during osteoclastogenesis. Knock-down of ApoE using a specific siRNA promoted the RANKL-mediated induction of osteoclast differentiation. While ApoE did not affect the activation of ERK, JNK, and p38 MAPK signaling pathways by RANKL, the phosphorylation of p65 trans-activation domain on serine 536 and transcription activity of NF-κB were reduced by ApoE overexpression. These findings suggest that ApoE plays an inhibitory role in osteoclast differentiation via the suppression of RANKL-dependent activation of NF-κB and induction of c-Fos and NFATc1.

Non-secretory multiple myeloma expressed as multiple extramedullary plasmacytoma with an endobronchial lesion mimicking metastatic cancer: A case report.

BACKGROUND: Non-secretory multiple myeloma (MM) is a rare condition that accounts for only 3% of MM cases and is defined by normal serum and urine immunofixation and a normal serum free light chain ratio. Non-secretory MM with multiple extramedullary plasmacytomas derived from endobronchial lesions is extremely rare and can be misdiagnosed as metastasis of solid cancer. CASE SUMMARY: A 36-year-old man presented with progressive facial swelling and nasal congestion with cough. Various imaging studies revealed an endobronchial mass in the left bronchus and a large left maxillary mass with multiple destructive bone metastatic lesions. He initially presented with lung cancer and multiple metastases. However, pathologic reports showed multiple extramedullary plasmacytomas in the left maxilla and the left bronchus. There was no change in the serum and urine monoclonal protein levels, and no abnormalities were observed in laboratory examinations, including hemoglobin, calcium, and creatinine levels. The bone marrow was hypercellular, with 13.49% plasma cells. The patient was diagnosed with non-secretory MM expressed as multiple extramedullary plasmacytomas with endobronchial lesions in a rare location. Radiation therapy for symptomatic lesions with high-dose dexamethasone was started, and the size of the left maxillary sinus lesion dramatically decreased. In the future, chemotherapy will be administered to control lesions in other areas. CONCLUSION: We present a rare case of non-secretory MM with multiple extramedullary plasmacytoma with an endobronchial lesion.

Myeloid-Specific PTP1B Deficiency Attenuates Inflammation-Induced and Ovariectomy-Induced Bone Loss in Mice by Inhibiting Osteoclastogenesis.

The differentiation and activity of bone-resorbing osteoclasts are tightly regulated to maintain the homeostasis of healthy bones. In this study, the role of protein tyrosine phosphatase 1B (PTP1B) during osteoclastogenesis was studied in myeloid-specific Ptpn1-deficient (conditional knockout [cKO]) mice. The mRNA and protein expression of PTP1B increased during the formation of mature osteoclasts from mouse bone macrophages on stimulation with macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL). The Ptpn1 cKO mice exhibited increased femoral trabecular bone volume with a decreased number and activity of osteoclasts compared with control mice. The in vitro culture of osteoclast precursors corroborated the inhibition of osteoclastogenesis in cKO cells compared with control, with concomitantly decreased RANKL-dependent proliferation, lower osteoclast marker gene expression, reduced nuclear expression of nuclear factor of activated T cells cytoplasmic 1 (NFATc1), diminished intracellular Ca2+ oscillations, and increased phosphorylation of proto-oncogene tyrosine-protein kinase Src on inhibitory tyrosine residue. In a ligature-induced periodontitis model, Ptpn1 cKO mice exhibited attenuated osteoclastogenesis and alveolar bone loss following the induction of inflammation. The Ptpn1-deficient mice were similarly protected from ovariectomy-induced bone loss compared with control mice. These results provide a novel regulatory role of PTP1B in osteoclastogenesis and suggest a potential as a therapeutic target for bone-lytic diseases. © 2021 American Society for Bone and Mineral Research (ASBMR).

Clinical outcome of pathologic Stage IIA endometrial adenocarcinoma after intravaginal brachytherapy alone.

PURPOSE: We studied the impact of different prognostic factors on the clinical outcome for the patients with pathologic Stage IIA endometrial adenocarcinoma who had surgical staging (SS) and received adjuvant high-dose-rate intravaginal brachytherapy (IVB) alone. METHODS AND MATERIALS: Sixty-one patients with Stage IIA endometrial adenocarcinoma were retrospectively studied. Cox proportional hazards regression was used to study prognostic factors. RESULTS: All the patients underwent SS between July 1994 and December 2005. The median age was 64 years (range, 46-71 years). The median number of lymph nodes sampled was 8 (range, 7-12). All the patients received adjuvant IVB to doses of 35-36Gy in four to five fractions prescribed to the surface. The myometrial invasion was <50% in 33 patients and > or =50% for 28 patients. The lymphovascular invasion (LVI) and the lower uterine segment involvement were identified in 18% and 61%, respectively. At a median followup of 64 months (range, 8-153 months), there were 7 patients who developed recurrences. On univariate analysis, the only factor significantly predictive for locoregional recurrence was LVI (p=0.01). In regard to overall survival (OS), factors that were significantly predictive on univariate analysis were LVI (p=0.03), tumor grade (p=0.04), and depth of myometrial invasion (p=0.04). The 5-year rates of vaginal and pelvic recurrences were 1.7% and 8.2%, respectively. The 5-year local control and OS rates were both 87%. CONCLUSIONS: Our results suggest excellent local control with adjuvant IVB alone for selected patients with Stage IIA endometrial adenocarcinoma. The patients with positive LVI and deep myometrial invasion have a worse locoregional control and OS despite SS and adjuvant IVB.

WDR76 is a RAS binding protein that functions as a tumor suppressor via RAS degradation.

Stability regulation of RAS that can affect its activity, in addition to the oncogenic mutations, occurs in human cancer. However, the mechanisms for stability regulation of RAS involved in their activity and its roles in tumorigenesis are poorly explored. Here, we identify WD40-repeat protein 76 (WDR76) as one of the HRAS binding proteins using proteomic analyses of hepatocellular carcinomas (HCC) tissue. WDR76 plays a role as an E3 linker protein and mediates the polyubiquitination-dependent degradation of RAS. WDR76-mediated RAS destabilization results in the inhibition of proliferation, transformation, and invasion of liver cancer cells. WDR76-/- mice are more susceptible to diethylnitrosamine-induced liver carcinogenesis. Liver-specific WDR76 induction destabilizes Ras and markedly reduces tumorigenesis in HRasG12V mouse livers. The clinical relevance of RAS regulation by WDR76 is indicated by the inverse correlation of their expressions in HCC tissues. Our study demonstrates that WDR76 functions as a tumor suppressor via RAS degradation.

Transglutaminase 2 regulates osteoclast differentiation via a Blimp1-dependent pathway.

Transglutaminase 2 (TG2) performs multiple reactions, including transamidation, and also plays a role in signal transduction as a GTP-binding protein. In this study, we reveal that TG2 controls osteoclast differentiation and bone homeostasis in mice. Osteoclasts specifically expressed the TG2 isoform among eight TG family members. Suppression in TG2 expression with siRNA led to increased osteoclast formation from primary mouse precursor cells in response to receptor activator of nuclear factor kappaB ligand (RANKL). This osteoclastogenic effect of TG2 knockdown was associated with enhanced induction of c-Fos and NFATc1 by RANKL. Moreover, TG2 knockdown up-regulated B lymphocyte-induced maturation protein 1 (Blimp1), which represses anti-osteoclastogenic genes, in a manner dependent on the NF-κB signaling pathway. To the contrary, TG2 overexpression inhibited osteoclast formation and the expression of osteoclastogenic genes. Consistent with these in vitro results, TG2 knockout mice exhibited lower trabecular bone mass and increased number of osteoclasts compared with wild-type mice. Taken together, our results provide strong evidence that TG2 plays an important role in bone metabolism by suppressing excessive osteoclastogenesis via the regulation of the NF-κB-Blimp1 signaling pathway.

Calcium-phosphorus product concentration is a risk factor of coronary artery disease in metabolic syndrome.

BACKGROUND: High serum phosphorus and the calcium-phosphorus product concentration has been associated with increased mortality and cardiovascular events in patients with chronic kidney disease. OBJECTIVE: This study was designed to determine the relationship between calcium-phosphorus product concentration and the presence of coronary artery calcification in subjects with metabolic syndrome (MetS). METHODS: We reviewed the medical records of 2056 general subjects with a mean age of 55.1 ± 9.9 years and a glomerular filtration rate of 88.9 ± 16.2 mL/min/1.73 m(2). The enrolled subjects consisted of 384 (18.7%) subjects with MetS and 1672 (81.3%) subjects without MetS. The severity of coronary artery calcification was assessed by the coronary artery calcification score (CACS) using multi-detector computed tomography (MDCT). RESULTS: The CACS correlated with calcium-phosphorus product concentration in subjects with MetS (r = 0.184, P < 0.01). The odds ratio of calcium-phosphorus product concentration having CACS >50 was 1.053 in subjects with MetS (P < 0.05). After adjustment for age, sex, diabetes, and dyslipidemia, calcium-phosphorus product concentrations had a positive correlation with CACS in subjects with MetS. In single regression analysis, calcium-phosphorus product concentration as independent variable was the significant predictor of CACS in subjects with MetS. Using a multivariate analysis, calcium-phosphorus product concentration remained a significant factor associated with CACS in subjects with MetS. CONCLUSIONS: Calcium-phosphorus product concentration was weakly associated with CACS and an independent factor predicting for CACS by MDCT in subjects with MetS. These results suggest that calcium-phosphorus product concentration might be considered as a risk factor of coronary artery disease in subjects with MetS.

Lipoproteins are an important bacterial component responsible for bone destruction through the induction of osteoclast differentiation and activation.

Bacterial infection can cause inflammatory bone diseases accompanied by the bone destruction resulting from excess generation of osteoclasts. Although lipoproteins are one of the major immunostimulating components of bacteria, little is known about their effects on bone metabolism. In this study, we investigated the role of lipoproteins in bacteria-induced bone destruction using Staphylococcus aureus wild type, its lipoprotein-deficient mutant, and synthetic lipopeptides Pam2CSK4 and Pam3CSK4 known to mimic bacterial lipoproteins. Formaldehyde-inactivated S. aureus or the synthetic lipopeptides induced severe bone loss in the femurs of mice after intraperitoneal administration and in a calvarial bone implantation model, whereas the lipoprotein-deficient S. aureus did not show such effects. Mechanism studies further identified three action mechanisms for the lipopeptide-induced osteoclast differentiation and bone resorption via (i) enhancement of osteoclast differentiation through Toll-like receptor 2 and MyD88-dependent signaling pathways; (ii) induction of pro-inflammatory cytokines, TNF-α and IL-6; and (iii) upregulation of RANKL expression with downregulation of osteoprotegerin expression in osteoblasts. Taken together, these results suggest that lipoprotein might be an important bacterial component responsible for bone destruction during bacterial infections through augmentation of osteoclast differentiation and activation.

Novel extraneural role of neurite outgrowth inhibitor A: modulation of osteoclastogenesis via positive feedback regulation of nuclear factor of activated T cell cytoplasmic 1.

Osteoclasts are bone-resorbing cells differentiated from macrophage/monocyte lineage precursors upon receptor activator of NF-κB ligand (RANKL) stimulation. In a proteomic approach to identify proteins involved in osteoclastogenesis, we observed a dramatic increase in the expression of neurite outgrowth inhibitor A (Nogo-A) upon RANKL stimulation of mouse bone marrow macrophages (BMMs) in a nuclear factor of activated T cell cytoplasmic 1 (NFATc1)-dependent manner. The knockdown of Nogo-A in BMMs significantly reduced RANKL-dependent osteoclast differentiation accompanied by diminished NFATc1 induction, suggesting that a positive feedback mechanism is involved. Conversely, Nogo-A overexpression in BMMs as well as in RAW264.7 macrophages greatly augmented osteoclastogenesis, with concomitant increase in the NFATc1 induction. Both the mitogen-activated protein kinase (MAPK) pathway and calcium oscillation, which are central to RANKL-dependent NFATc1 activation and induction, were enhanced by Nogo-A. Finally, Nogo-A knockdown in mouse calvariae prevented interleukin 1 (IL-1)-induced bone loss. These findings not only reveal an unprecedented extraneural role of Nogo-A in osteoclastogenesis but also suggest a novel drug target against bone-lytic diseases.

Misexpression of Dickkopf-1 in endothelial cells, but not in chondrocytes or hypertrophic chondrocytes, causes defects in endochondral ossification.

Developing cartilage serves as a template for long-bone development during endochondral ossification. Although the coupling of cartilage and bone development with angiogenesis is an important regulatory step for endochondral ossification, the molecular mechanisms are poorly understood. One possible mechanism involves the action of Dickkopf (DKK), which is a family of soluble canonical Wnt antagonists with four members (DKK1-4). We initially observed opposite expression patterns of Dkk1 and Dkk2 during angiogenesis and chondrocyte differentiation: downregulation of Dkk1 and upregulation of Dkk2. We examined the in vivo role of Dkk1 and Dkk2 in linking cartilage/bone development and angiogenesis by generating transgenic (TG) mice that specifically express Dkk1 or Dkk2 in chondrocytes, hypertrophic chondrocytes, or endothelial cells. Despite specific expression pattern during cartilage development, chondrocyte- and hypertrophic chondrocyte-specific Dkk1 and Dkk2 TG mice showed normal developmental phenotypes. However, Dkk1 misexpression in endothelial cells resulted in defects of endochondral ossification and reduced skeletal size. The defects are caused by the inhibition of angiogenesis in developing bone and subsequent inhibition of apoptosis of hypertrophic chondrocytes and cartilage resorption.

PlexinA2 mediates osteoblast differentiation via regulation of Runx2.

The imbalance between bone-resorbing osteoclasts and bone-forming osteoblasts often leads to bone destructive diseases such as osteoporosis. In contrast to the development of several antiresorptive agents for osteoporosis therapy, discovery of anabolic drugs has been difficult because of an insufficient understanding of the complex mechanism of bone formation. In a microarray analysis with mouse preosteoblast cells, we found that PlexinA2 (PlxnA2), a molecule previously known to mediate axon guidance in neural development, was upregulated by the osteogenic factor BMP2. PlxnA2-specific siRNA decreased Runx2 expression, osteoblast differentiation, and mineralization. Runx2 overexpression restored osteoblastic differentiation of PlxnA2-knockdown cells. PlxnA2 was associated with both type 1 and 2 BMP receptors, and BMP2 increased the interaction between PlxnA2 and type 1 receptors. PlxnA2 also affected Smad and Akt signaling pathways downstream of BMP2. Taken together, the results of our study reveal that PlxnA2 has a pro-osteogenic function by modulating BMP2 signaling. Therefore, PlxnA2 may be a useful target for development of bone anabolic therapeutics.

Correlation between N-Terminal Pro-Brain Natriuretic Peptide and Doppler Echocardiographic Parameters of Left Ventricular Filling Pressure in Atrial Fibrillation.

BACKGROUND: Atrial fibrillation (AF) is the most common cardiac arrhythmia with a population prevalence of about 1%. Natriuretic peptide level is elevated in patients with AF with diastolic dysfunction even with a normal left ventricular (LV) ejection fraction. The N-terminal pro-brain natriuretic peptide (NT-proBNP) level and Doppler echocardiographic parameters for diastolic function have shown correlation with LV filling pressures. We aimed to evaluate the relationship between echocardiographic parameters and serum NT-proBNP in patients with AF with preserved LV ejection fraction. METHODS: We examined transthoracic echocardiography and NT-proBNP levels in the patients with AF and patients with sinus rhythm. Blood samples were taken for serum NT-proBNP measurements within 24 hours of echocardiographic examination. The group 1 was the patients with sinus rhythm (n = 30, mean age 68 ± 13 years) and the group 2 was the patients with AF (n = 33, mean age 70 ± 14 years). RESULTS: The group 2 patients had significantly higher mitral E, E' (lateral annulus), E/E' (septal annulus), left atrial (LA) volume index, LA size, pulmonary vein diastolic velocity, and NT-proBNP level than those of group 1 patients (p < 0.05). The area under the receiver-operating characteristic curve showed a NT-proBNP had good diagnostic power for E/E' (septal annulus) > 15 in patients with AF at cutoff value of 433 pg/mL. CONCLUSION: NT-proBNP level is well correlated with Doppler echocardiographic parameters of diastolic function in patients with AF and preserved LV ejection fraction. NT-proBNP level more than 433 pg/mL may suggest elevated LV filling pressure in patients with AF.

The primary patency of percutaneous transluminal angioplasty in hemodialysis patients with vascular access failure.

BACKGROUND AND OBJECTIVES: Dysfunction of arteriovenous fistulas (AVFs) and arteriovenous grafts (AVGs) contributes significantly to morbidity and hospitalization in the dialysis population. We evaluated the primary patency of AVFs following percutaneous transluminal angioplasty (PTA) in haemodialysis patients. SUBJECTS AND METHODS: We performed 231 interventions in 118 patients with a mean age of 62.1±12.9 years. We performed 122 interventions in 53 AVG patients (44.9%), and 109 interventions in 65 AVF patients (55.1%). If there was thrombosis of the vascular access, urokinase was administered and/or thrombus aspiration was performed. The stent was inserted when balloon dilatation did not expand sufficiently or elastic recoil occurred. RESULTS: For the 118 patients, the median patency time was 10.45±10.29 months at 92 months of follow-up. The primary patencies for stenotic AVFs at 6, 12, 24, 36, 48, and 60 months were 63.4%, 41.4%, 17.0%, 9.7%, 7.3%, and 2.4%, respectively. The primary patencies for AVGs at 6, 12, 24, and 36 months were 36.9%, 19.5%, 10.8%, 2.1%, respectively, and were obtained by means of the Kaplan-Meier analysis (log rank=6.42, p<0.05). The median patency time was 11.0 months and 4.45 months in the non-thrombus and thrombus groups, respectively. The complication rate was 1.73% (4/231); two cases of pseudoaneurysms and two cases of extravasation were detected. All therapy failures (5/231) occurred in thrombotic lesions of AVGs and were treated surgically. CONCLUSION: PTA is an efficacious method for the correction of stenosis of AVFs for hemodialysis, thus prolonging the patency of the fistulas.

A case of staphylococcal tricuspid valve endocarditis with para-aortic abscess in a patient with bicuspid aortic valve.

Paravalvular abscess is a serious complication of infective endocarditis. The aortic valve and its adjacent ring are more susceptible to abscess formation and paravalvular extension than the mitral valve. A 15-years old patient with bicuspid aortic valve presented with staphylococcal tricuspid valve endocarditis complicated by para-aortic abscess that ruptured into the aortic sinus. We report the clinical, laboratory and echocardiographic features and treatment of this patient and conduct a literature review on this subject.

A novel role of dipeptidyl peptidase 9 in epidermal growth factor signaling.

Dipeptidyl peptidase IV (DPP4), DPP8, DPP9, and fibroblast activation protein (FAP), the four proteases of the DPP4 gene family, have unique peptidase and extra-enzymatic activities that have been implicated in various diseases including cancers. We report here a novel role of DPP9 in regulating cell survival and proliferation through modulating molecular signaling cascades. Akt (protein kinase B) activation was significantly inhibited by human DPP9 overexpression in human hepatoma cells (HepG2 and Huh7) and human embryonic kidney cells (HEK293T), whereas extracellular signal-regulated kinases (ERK1/2) activity was unaffected, revealing a pathway-specific effect. Interestingly, the inhibitory effect of DPP9 on Akt pathway activation was growth factor dependent. DPP9 overexpression caused apoptosis and significantly less epidermal growth factor (EGF)-mediated Akt activation in HepG2 cells. However, such inhibitory effect was not observed in cells stimulated with other growth factors, including connective tissue growth factor, hepatic growth factor, insulin or platelet-derived growth factor-BB. The effect of DPP9 on Akt did not occur when DPP9 enzyme activity was ablated by either mutagenesis or inhibition. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is a major downstream effector of Ras. We found that DPP9 and DPP8, but not DPP4 or FAP, associate with H-Ras, a key signal molecule of the EGF receptor signaling pathway. These findings suggest an important signaling role of DPP9 in the regulation of survival and proliferation pathways.