In vitro and in vivo detection of tunneling nanotubes in normal and pathological osteoclastogenesis involving osteoclast fusion.

Osteoclasts are multinucleated cells formed through specific recognition and fusion of mononuclear osteoclast precursors derived from hematopoietic stem cells. Detailed cellular events concerning cell fusion in osteoclast differentiation remain ambiguous. Tunneling nanotubes (TNTs), actin-based membrane structures, play an important role in intercellular communication between cells. We have previously reported the presence of TNTs in the fusion process of osteoclastogenesis. Here we analyzed morphological details of TNTs using scanning electron microscopy. The osteoclast precursor cell line RAW-D was stimulated to form osteoclast-like cells, and morphological details in the appearance of TNTs were extensively analyzed. Osteoclast-like cells could be classified into three types; early osteoclast precursors, late osteoclast precursors, and multinucleated osteoclast-like cells based on the morphological characteristics. TNTs were frequently observed among these three types of cells. TNTs could be classified into thin, medium, and thick TNTs based on the diameter and length. The shapes of TNTs were dynamically changed from thin to thick. Among them, medium TNTs were often observed between two remote cells, in which side branches attached to the culture substrates and beaded bulge-like structures were often observed. Cell-cell interaction through TNTs contributed to cell migration and rapid transport of information between cells. TNTs were shown to be involved in cell-cell fusion between osteoclast precursors and multinucleated osteoclast-like cells, in which movement of membrane vesicles and nuclei was observed. Formation of TNTs was also confirmed in primary cultures of osteoclasts. Furthermore, we have successfully detected TNTs formed between osteoclasts observed in the bone destruction sites of arthritic rats. Thus, formation of TNTs may be important for the differentiation of osteoclasts both in vitro and in vivo. TNTs could be one target cellular structure for the regulation of osteoclast differentiation and function in bone diseases.

Modulation of osteoclastogenesis through adrenomedullin receptors on osteoclast precursors: initiation of differentiation by asymmetric cell division.

Adrenomedullin (ADM), a member of the calcitonin family of peptides, is a potent vasodilator and was shown to have the ability to modulate bone metabolism. We have previously found a unique cell surface antigen (Kat1 antigen) expressed in rat osteoclasts, which is involved in the functional regulation of the calcitonin receptor (CTR). Cross-linking of cell surface Kat1 antigen with anti-Kat1 antigen monoclonal antibody (mAbKat1) stimulated osteoclast formation only under conditions suppressed by calcitonin. Here, we found that ADM provoked a significant stimulation in osteoclastogenesis only in the presence of calcitonin; a similar biological effect was seen with mAbKat1 in the bone marrow culture system. This stimulatory effect on osteoclastogenesis mediated by ADM was abolished by the addition of mAbKat1. 125I-labeled rat ADM (125I-ADM)-binding experiments involving micro-autoradiographic studies demonstrated that mononuclear precursors of osteoclasts abundantly expressed ADM receptors, and the specific binding of 125I-ADM was markedly inhibited by the addition of mAbKat1, suggesting a close relationship between the Kat1 antigen and the functional ADM receptors expressed on cells in the osteoclast lineage. ADM receptors were also detected in the osteoclast progenitor cells in the late mitotic phase, in which only one daughter cell of the dividing cell express ADM receptors, suggesting the semiconservative cell division of the osteoclast progenitors in the initiation of osteoclastogenesis. Messenger RNAs for the receptor activity-modifying-protein 1 (RAMP1) and calcitonin receptor-like receptor (CRLR) were expressed in cells in the osteoclast lineage; however, the expression of RAMP2 or RAMP3 was not detected in these cells. It is suggested that the Kat1 antigen is involved in the functional ADM receptor distinct from the general ADM receptor, consisting of CRLR and RAMP2 or RAMP3. Modulation of osteoclastogenesis through functional ADM receptors abundantly expressed on mononuclear osteoclast precursors is supposed to be important in the fine regulation of osteoclast differentiation in a specific osteotrophic hormonal condition with a high level of calcitonin in blood.

Fusion imaging-guided radiofrequency ablation for residual hepatocellular carcinoma invisible on ultrasound after transcatheter arterial chemoembolization.

PURPOSE: This study aimed to investigate the technical efficiency and therapeutic response of fusion imaging (considered as virtual navigation) between contrast-enhanced ultrasound (CEUS) and contrast-enhanced computed tomography/magnetic resonance imaging (CECT/CEMRI) for the guidance of radiofrequency ablation (RFA) in patients with residual hepatocellular carcinoma (HCC) after transcatheter arterial chemoembolization (TACE). METHODS: For this prospective study, 98 patients with residual HCC lesions after TACE treatment were enrolled between June 2017 and December 2020. All the lesions were invisible on conventional ultrasound scans. Percutaneous RFA was performed using either CEUS (CEUS group, 52 lesions) or virtual navigation (VN group, 46 lesions) guidance. The lesion display rate, disease-free survival rate, local recurrence rate, overall survival rate and complication incidence were calculated and compared. RESULTS: Fusion imaging had a significant impact on the RFA outcomes (hazard ratio, 2.629; 95% confidence interval, 1.256-5.505; p = .01). The median disease-free survival time of the VN group was significantly higher than that of the CEUS group (10.9 vs. 8.8 months; p = .007). The local recurrence rates after 3, 6 and 12 months in the VN group were significantly lower than those in the CEUS group (p = .014, .002 and .011). The minor complication rate was not significantly different between the two groups. CONCLUSIONS: CEUS-CECT/CEMRI fusion imaging for guiding RFA enables an efficient and useful therapy of inconspicuous HCC lesions after TACE. The novel solution prolongs the disease-free survival time and reduces the long-term local recurrence of residual lesions treated when using virtual-navigation (VN)-guided RFA.

Endoscopic Ultrasonography-Guided Versus Percutaneous Drainage for the Recurrent Pancreatic Fluid Collections.

BACKGROUND Ultrasonography-guided percutaneous drainage for pancreatic fluid collections is associated with a high recurrence rate and endoscopic ultrasonography (EUS)-guided drainage is a valuable approach. Our aim was to compare the efficacy and safety of percutaneous and EUS-guided drainage for the recurrent pancreatic fluid collections. MATERIAL AND METHODS A retrospective analysis of percutaneous-guided and EUS-guided procedures for pancreatic fluid collections drainages at a single tertiary care center between February 2017 and May 2018 was performed. Treatment success, adverse events, recurrence, need for surgery, length of hospital stays, and number of follow-up computed tomography (CT) scan were assessed. RESULTS A total of 119 pancreatic fluid collections treated with initial percutaneous drainage were included in this study and 35 patients had recurrent pancreatic fluid collections. Recurrent patients were classified based on drainage method: EUS-guided drainage (18 patients) and the second percutaneous drainage (17 patients). EUS-guided drainage revealed a shorter length of hospital stays (P<0.001), less re-intervention (P=0.047), fewer number of follow-up CT scans (P=0.006) compared with the initial percutaneous drainage. Furthermore, we also compared the clinical outcomes between the EUS-guided drainage and the second percutaneous drainage for the recurrent PFC after initially failed percutaneous drainage. EUS-guided drainage showed higher clinical success (P=0.027), shorter length of hospital stays (P<0.001), less re-intervention (P=0.012), fewer number of follow-up CT scan (P<0.001) and less recurrence P=0.027) compared to the second percutaneous drainage procedure. CONCLUSIONS EUS-guided drainage is an effective and appropriate method to treat the recurrent pancreatic fluid collections after initially failed percutaneous drainage procedure, with the advantage of higher clinical success, shorter length of hospital stays, less re-intervention, fewer number of follow-up CT scan and less recurrence compared to the percutaneous drainage.

Usefulness of new shear wave elastography in early predicting the efficacy of neoadjuvant chemotherapy for patients with breast cancer: where and when to measure is optimal?

BACKGROUND: The aim of this study was to investigate the diagnosis performance of new shear wave elastography (sound touch elastography, STE) in the prediction of neoadjuvant chemotherapy (NAC) response at an early stage in breast cancer patients and to determine the optimal measurement locations around the lesion in different ranges. METHODS: One hundred and eight patients were analyzed in this prospective study from November 2018 to December 2020. All patients completed NAC treatment and underwent STE examination at three time points [the day before NAC (t0); the day before the second course (t1); the day before third course (t2)]. The stiffness of the whole lesion (G), 1-mm shell (S1) and 2-mm shell (S2) around the lesion was expressed by STE parameters. The relative changes (∆stiffness) of STE parameters after the first and second course of NAC were calculated and shown as the variables [Δ(t1) and Δ(t2)]. The diagnostic accuracy of STE was evaluated by means of receiver operating characteristic curve analysis. RESULTS: The ∆stiffness (%) including ∆Gmean(t2), ∆S1mean(t2) and ∆S2mean(t2) all showed significant differences between pathological complete response (pCR) and non-pCR groups. ∆S2mean(t2) displayed the best predictive performance for pCR (AUC = 0.842) with an ideal ∆stiffness threshold value - 26%. CONCLUSIONS: Measuring the relative changes in the stiffness of surrounding tissue or entire lesion with STE holds promise for effectively predicting the response to NAC at its early stage for breast cancer patients and ∆stiffness of shell 2 mm after the second course of NAC may be a potential prediction parameter.

Quantitative Ultrasound Elastography Methods in Focal Liver Lesions Including Hepatocellular Carcinoma: From Diagnosis to Prognosis.

ABSTRACT: The ability of ultrasound elastography to diagnose focal liver lesions and determine their prognoses including hepatocellular carcinoma (HCC) is unclear. At present, radiofrequency ablation and liver resection are the most common treatments for HCC. However, the survival rate remains disappointing because of recurrences and postoperative liver failure, necessitating the development of noninvasive approaches. There is currently no systematic definition of an elastic technique for measuring liver stiffness to predict the recurrence of HCC after radiofrequency ablation and postoperative liver failure. In this review, recent advances in ultrasound elastography for the diagnosis and prognosis of focal liver lesions are discussed including HCC.

Assessment of liver fibrosis with liver and spleen stiffness measured by sound touch elastography, serum fibrosis markers in patients with chronic hepatitis B.

OBJECTIVES: To evaluate the performance of liver stiffness (LS) and spleen stiffness (SS) by using the sound touch elastography (STE) technique and compare with those of the splenic index, aspartate transaminase-to-platelet ratio index (APRI), fibrosis-4 (FIB-4) index, King's score and combined models for diagnosing and staging fibrosis in chronic hepatitis B (CHB). METHODS: One hundred patients with CHB underwent STE and serological tests. LS and SS values were measured with STE technique, and splenic index was calculated. Staging of fibrosis was determined with liver biopsy. Correlations between the individual parameters and the stage of fibrosis were evaluated with the Spearman correlation analysis. The area under the receiver operating characteristic curve (AUROC) was calculated to analyze the performance of all methods. RESULTS: Among all individual parameters, LS showed the highest AUROC for diagnosing fibrosis of ≥S2, ≥S3, and S4 stages (AUROC: 0.70, 0.86, and 0.96, respectively; all P < 0.05). The AUROC of combined model 1 (LS and SS) and 2 (LS, SS, APRI, FIB-4 index, King's score) for diagnosing ≥S2, ≥S3, and S4 fibrosis were 0.70, 0.86, 0.97, and 0.70, 0.86, 0.96, respectively, which were higher than those of APRI, FIB-4 index and the King's score (P < 0.05). No significant differences were found between two combined models and LS for staging fibrosis (P > 0.05). CONCLUSIONS: LS measurement is reliable for diagnosing and staging fibrosis in CHB, with a better performance than SS, splenic index and serum biomarkers. It is also comparable with the performance of combined models.

Replacement Process of Carbonate Apatite by Alveolar Bone in a Rat Extraction Socket.

The objective of this study was to investigate a bone graft substitute containing carbonate apatite (CO3Ap) to analyze bone replacement and the state of bone formation in vitro and in vivo compared with autogenous bone (AB) or control. An osteoclast precursor cell line was cultured with AB or CO3Ap, and morphological analysis using scanning electron microscopy and a tartrate-resistant acid phosphatase activity assay were performed. The right maxillary first and second molars of Wistar rats were extracted and compensated by AB or CO3Ap granules. Following implantation, the bone formation state was evaluated after 3, 5, 7, 14, and 28 days of surgery by micro-computed tomography and immunohistostaining. The osteoclast-like cell morphology was typical with many cell protrusions in the AB and CO3Ap groups. Additionally, the number of osteoclast-like cells formed in the culture increased in each group; however, there was no significant difference between the AB and CO3Ap groups. Five days after tooth extraction, osteoclasts were observed near CO3Ap. The bone thickness in the CO3Ap group was significantly increased than that in the control group and the bone formation in the CO3Ap group increased by the same level as that in the AB group. CO3Ap is gradually absorbed by osteoclasts in the extraction socket and is easily replaced by alveolar bone. The process of bone replacement by osteoclasts is similar to that of autologous bone. By observing the process of bone replacement in more detail, it may be possible to gain a better understanding of the bone formation and control the amount of bone after surgery.

Phospho-Smad3L promotes progression of hepatocellular carcinoma through decreasing miR-140-5p level and stimulating epithelial-mesenchymal transition.

BACKGROUND: The transforming growth factor ß (TGF-ß) activates JNK, phosphorylates Smad3 to linker-phosphorylated Smad3 (pSmad3L), resulting in liver tumorigenesis. However, the effect of pSmad3L on hepatocellular carcinoma (HCC) prognosis is obscure. AIM: To detect the effect of pSmad3L on HCC prognosis and investigate the mechanism. METHODS: The expressions of pSmad3L, E-cadherin, vimentin and MicroRNA-140-5p (miR-140-5p) were detected by using immunohistochemistry, immunofluorescence and in situ hybridization. Next, the relationships of pSmad3L and HCC patients' prognoses, pSmad3L and EMT markers, pSmad3L and miR-140-5p were analyzed using Spearman's rank correlation test. JNK/pSmad3L specific inhibitor SP600125 or Smad3 mutant plasmid was used to suppress JNK/pSmad3L pathway, and QPCR assay was performed to investigate the effect of pSmad3L on miR-140-5p level. The proliferation and invasion of hepatoma cells were observed using colony formation assay and transwell assay. RESULTS: We demonstrated that patient with high level of pSmad3L predicted poor prognosis. Next, we verified that pSmad3L promoted EMT of hepatoma cells in vivo and in vitro. In order to investigate the mechanism, we verified a negative correlation between pSmad3L and miR-140-5p, which was an EMT inhibitor, in the liver tissues of HCC patient and diethylnitrosamine (DEN)-induced rat HCC model. We further used SP600125 or pSmad3L mutant plasmid to decrease pSmad3L level of hepatoma cells, and inhibition of pSmad3L increased miR-140-5p level and suppressed EMT of hepatoma cells. CONCLUSIONS: JNK/pSmad3L pathway induces EMT by inhibiting miR-140-5p in HCC progression.

Genome-Based Analysis of a Sequence Type 1049 Hypervirulent Klebsiella pneumoniae Causing Bacteremic Neck Abscess.

Hypervirulent Klebsiella pneumoniae (hvKP) has raised grave concerns in recent years and can cause severe infections with diverse anatomic locations including liver abscess, meningitis, and endophthalmitis. However, there is limited data about neck abscess caused by hvKP. A K. pneumoniae strain Kp_whw was isolated from neck abscess. We characterized the genetic background, virulence determinates of the strain by genomic analysis and dertermined the virulence level by serum resistance assay. Kp_whw belonged to sequence type (ST) 1049 K locus (KL) 5. Kp_whw showed hypermucoviscosity phenotype and was resistant to ampicillin but susceptible to the majority of the other antimicrobial agents. A pLVPK-like virulence plasmid and a chromosomal ICEKp5-like mobile genetic element were carried by Kp_whw, resulting in the risk of dissemination of hypervirulence. The strain exhibited relative higher level of core genome allelic diversity than accessory genome profile, in comparison to hvKP of K1/K2 serotype. Kp_whw was finally demonstrated as virulent as the ST23 K1 serotype hvKP strain NTUH-K2044 in vitro. In conclusion, this work elaborates the genetic background of a clinical hvKP strain with an uncommon ST, reinforcing our understanding of virulence mechanisms of hvKP.

Pre-operative Detection of Liver Fibrosis in Hepatocellular Carcinoma Patients Using 2D Shear Wave Elastography: Where to Measure?

The aim of this study was to pre-operatively investigate the diagnostic performance of 2D shear wave elastography (2D-SWE) for staging liver fibrosis and inflammation in patients with hepatocellular carcinoma (HCC) who then undergo surgery and to determine the optimal locations for measurement. In total, 106 patients were enrolled in this prospective study from March 2017 to May 2018. Two-dimensional SWE was used to measure liver stiffness (LS) in each patient 0-1, 1-2 and 2-5 cm from the tumor border (groups 1, 2 and 3, respectively). Spearman's correlation was used to evaluate the relationships between LS and hepatic fibrosis and between LS and inflammation. Receiver operating characteristic curve (ROC) analysis was used to evaluate the diagnostic accuracy of 2D-SWE. The technical success rate of SWE in tissue distant from the tumor (group 3) was significantly higher than that in peri-tumoral tissue (groups 1 and 2) (p < 0.001). Moreover, the area under the ROC for diagnosing cirrhosis (F4) and severe inflammation (A3) was higher for group 3 than for groups 1 and 2. Our results suggest that 2D-SWE is a helpful approach to assessment of hepatic fibrosis in HCC patients before hepatic resection. We found that to achieve a superior success rate and preferable diagnosis accuracy for patients with HCC, LS measurement should be performed 2-5 cm from the tumor margin.

[The clinical observation of bipolar transurethral plasma kinetic resection of prostate for high risk benign prostate hyperplasia].

OBJECTIVE: To evaluate therapeutic effect and reliability of bipolar transurethral plasma kinetic prostatectomy (TUPKP) for high risk level benign prostatic hyperplasia (BPH). METHODS: A total of 230 cases of high risk of BPH were treated with TUPKP. Among them, 132 cases with the residual urine of 40 to 420 ml had accepted long term but inefficient medical therapy, 98 cases were suffered with repeating acute urinary retention. One hundred and seventy-three cases with the functional capacity>4 MET were performed the standard transurethral resection of the prostate (TURP), the other 57 cases with the functional capacity<4 MET were accepted the minimally invasive TURP. Among them 12 cases complicated with bladder stones accepted Ho: YAG lithotripsy priory. The international prostate symptom score (IPSS), The maximal urinary flow rate (Qmax) and residual urine of the 2 groups before and after operation were analyzed. RESULTS: There was no transurethral resection syndrome occurred in both groups. After 3 to 12 months of follow-up postoperatively, the IPSS of the two groups were reduced from (21.9+/-5.7) and (23.7+/-5.0) to (4.4+/-2.3) and (5.5+/-2.4), residual urine were reduced from (61.8+/-18.4) ml and (103.9+/-77.3) ml to (13.0+/-6.2) ml and (15.8+/-6.1) ml, respectively. The Qmax was increased from (5.7+/-3.0) ml/s and (4.8+/-2.8) ml/s to (20.9+/-6.3) ml/s and (16.8+/-3.9) ml/s, there existed significant differences (P<0.01). However the IPSS, Qmax and residual urine of the standard group had progressed more obviously than the minimally invasive TURP group (P<0.05). CONCLUSIONS: It is safe and effective to use TUPKP for treating high risk patients of BPH with classic TURP and minimally invasive TURP according to different functional capacity. When the functional capacity is more than 4 MET, the standard procedures is preferred.