Influence of rabbit notochordal cells on symptomatic intervertebral disc degeneration: anti-angiogenic capacity on human endothelial cell proliferation under hypoxia.

OBJECTIVES: Symptomatic degenerative disc disease (DDD) is associated with neovascularization and nerve ingrowth into intervertebral discs (IVDs). Notochordal cells (NCs) are key cells that may lead to regeneration of IVDs. However, their activities under conditions of hypoxia, the real environment of IVD, are not well known. We hypothesized that NCs may inhibit neovascularization by interacting with endothelial cells (ECs) under hypoxia. DESIGN: Human IVDs were isolated and cultured to produce nucleus pulposus (NP) cell conditioned medium (NPCM). Immortalized human microvascular ECs were cultured in NPCM with notochordal cell-rich rabbit nucleus pulposus cells (rNC) under hypoxia. Vascular endothelial growth factor (VEGF), vascular cell adhesion molecule (VCAM), and interleukin-8 (IL-8) were analyzed by ELISA. Focal adhesion kinase (FAK), filamentous actin (F-actin), and platelet-derived growth factor (PDGF) were evaluated to investigate EC activity. Wound-healing migration assays were performed to examine EC migration. RESULTS: The VEGF level of EC cells cultured in NPCM was significantly higher under hypoxia compared to normoxia. VEGF expression was significantly decreased, and FAK, F-actin, PDGF expression were inhibited when ECs were cocultured with rNCs under hypoxia. ECs cocultured with rNC in NPCM showed significantly decreased migratory activity compared to those without rNC under hypoxia. CONCLUSIONS: The angiogenic capacity of ECs was significantly inhibited by NCs under hypoxia via a VEGF-related pathway. Our results suggest that NCs may play a key role in the development of IVDs by inhibiting vascular growth within the disc, and this may be a promising novel therapeutic strategy for targeting vascular ingrowth in symptomatic DDD.

Whole-breast US following mammography and breast MRI in newly diagnosed breast cancer patients: can it be more than just a guidance tool for biopsy?

AIM: To evaluate the role of ultrasound (US) following magnetic resonance imaging (MRI) and mammography in patients with newly diagnosed breast cancers by assessing the additional cancer detection rate of US. MATERIAL AND METHODS: Two hundred and twenty-five women who had undergone 225 MRI examinations followed by US were included. An US-detected additional cancer was defined as a lesion detected using breast US that had not been detected by MRI, and which was shown to be malignant at histopathology. The rate of additional cancer detection, incidence of additional malignancies, positive predictive value (PPV), and false-positive (FP) rate were analysed. Factors associated with an increase in the additional cancer detection rate were analysed. RESULTS: The additional cancer detection rate was 0% (0/225) for the ipsilateral breast and 0.9% (2/225) for the contralateral breast, and the PPVs were 0% (0/5) and 100% (2/2), respectively. The overall TP:FP ratio was 0.4 (2:5). The additional cancer detection rate was higher for cases with moderate and severe background parenchymal enhancement than cases with minimal and mild background parenchymal enhancement (p=0.003). The additional cancer detection rate for cases with moderate and severe background parenchymal enhancement was 5.7% (2/35) for the contralateral breast (p=0.003). CONCLUSION: Preoperative breast US following MRI and mammography can help clinicians screen for contralateral cancers with an additional detection rate of 0.9%. Moreover, whole-breast US might be a useful contralateral screening modality in cases with moderate or marked parenchymal enhancement on breast MRI.

Adding MRI to ultrasound and ultrasound-guided fine-needle aspiration reduces the false-negative rate of axillary lymph node metastasis diagnosis in breast cancer patients.

AIM: To evaluate whether adding magnetic resonance imaging (MRI) to ultrasound (US) and US-guided fine-needle aspiration (US-FNA) can reduce the false-negative rate (FNR) in the diagnosis of axillary lymph node metastasis (ALNM) in breast cancer patients, and to assess false-negative diagnosis of N2 and N3 disease when adding MRI to US and US-FNA. MATERIALS AND METHODS: From March 2012 to February 2013, 497 breast cancer patients were included in the study. ALNM was evaluated according to US and US-FNA prior to MRI. Second-look US was performed when MRI showed positive findings of ALNM. If second-look US also revealed a positive finding, US-FNA was performed. Diagnostic performance, including FNR, was calculated for US and US-FNA with and without MRI. The negative predictive value (NPV) of N2 and N3 disease was evaluated in negative cases based on US and US-FNA with MRI. RESULTS: A total of 159 of 497 (32.0%) patients were found to have ALNM. Among them, 92 patients were diagnosed with metastasis on US and US-FNA. When adding MRI to US and US-FNA, an additional six patients were diagnosed with metastasis. The FNR of diagnosis of ALNM was improved by the addition of MRI (42.1% versus 38.4%, p = 0.0143). The NPV for N2 and N3 disease was 98% (391/399) based on US and US-FNA with MRI. CONCLUSION: Adding MRI to US and US-FNA could reduce the FNR of the diagnosis of ALNM. Furthermore, US and US-FNA with MRI may exclude 98% of N2 and N3 disease.

Temperature-dependent rheological property changes of thermoplastic gutta-percha root filling materials.

AIM: To compare the penetration resistance (PR) of six thermoplastic gutta-percha (GP) materials against needle penetration at different temperatures. METHODOLOGY: The PR of the following materials based on warm gutta-percha techniques, Beefill GP Cartridge (Beefill), GP Obturator-Hard/Medium/Soft (Dia-H, Dia-M, Dia-S), Obtura (Obtura) and Tactendo Gutta (Tactendo), was measured as a function of temperature change (from 30 °C to 200 °C). The PR was measured using a force gauge, mounted on a motor-driven micrometre stage, which pushed the penetration needle against GP in a glass cylinder at the designated temperatures. Data were analysed using Kruskal-Wallis anova and Duncan's multiple range tests at 5% significance. RESULTS: The mean PR values for all assessed materials ranged from 15.55 MPa to 26.02 MPa at 30 °C, decreased to 0.18-1.61 MPa at 60 °C, and finally reached 0.02-0.12 MPa at 200 °C. The Obtura had the highest PR values at most of the measured temperatures. At 40-60 °C, Obtura had the highest PR value (P < 0.05), and Tactendo had the lowest amongst the tested materials. At 70-200 °C, both the mean PR values and melt flow temperature (MFR) of Obtura, Dia-H and Dia-M were significantly higher than those of other materials (P < 0.05). CONCLUSIONS: The PR values were changed by both the heating temperature and the brand or type of GP materials tested. Each material was associated with drastic decreases before reaching 60 °C in different degrees, and Obtura had the highest PR at most of the measured temperatures.

Comparison of Cancer Yields and Diagnostic Performance of Screening Mammography vs. Supplemental Screening Ultrasound in 4394 Women with Average Risk for Breast Cancer.

PURPOSE: The effectiveness of supplemental screening ultrasound (US) was investigated in women ≥ 40 years at average risk for breast cancer regardless of breast parenchymal density. A total of 4394 women at average risk and having previously undergone screening mammography were classified as the mammography group.  MATERIALS AND METHODS: Of 4394 women, 2005 underwent screening US after a final assessment of category 1 or 2 on screening mammography, and were categorized as the US group. Category 0, 4, and 5 on mammography and 3, 4, and 5 on US were defined as positive. The cancer yields per 1000 women and diagnostic performance of two groups were compared. RESULTS: The total cancer and invasive cancer yields for the mammography group were 3.0 (95 % confidence interval 1.6, 5.1) and 2.0 (95 % CI, 0.9, 3.9) per 1000 women, higher than the US values of 2.0 (0.5, 5.1) and 1.0 (0.1, 3.6), not statistically significant. The specificity, accuracy, and positive predictive value (PPV) for mammography were 88.90 % (87.93, 89.81), 88.85 % (87.88, 89.76), and 2.61 % (1.39, 4.41), significantly higher than the US values of 69.07 % (66.99, 71.09), 69.13 % (67.05, 71.15), and 0.64 % (0.18, 1.64). The short-term follow-up rate of mammography was 5.51 % (4.85, 6.22), significantly lower than the rate of 26.58 (24.66, 28.58) for US.  CONCLUSION: Supplemental screening US in mammographically negative breasts can find additional carcinomas in women at average risk but is not as effective as screening mammography because of the lower cancer yield, invasive cancer yield, specificity, accuracy, PPV and a high short-term follow-up rate.

Effects of secreted factors in culture medium of annulus fibrosus cells on microvascular endothelial cells: elucidating the possible pathomechanisms of matrix degradation and nerve in-growth in disc degeneration.

OBJECTIVE: To test whether the interaction between annulus fibrosus cells (AFCs) and endothelial cells (ECs) disrupts matrix homeostasis and stimulates production of innervation mediators. METHODS: Human microvascular ECs were cultured in the conditioned media of AF cell culture derived from degenerated human surgical specimen. Matrix-metalloproteinases (MMPs) and platelet-derived growth factor (PDGF) of ECs of this culture were analyzed by qRT-PCR, Western, and immunofluorescence. Vascular endothelial growth factor (VEGF), Interleukin-8 (IL-8), and nerve growth factor (NGF) in the media of this cell culture were assayed by ELISA. To determine the effects of ECs on AFCs, qRT-PCR was performed to determine mRNA levels of collagen I, II and aggrecan in AFCs cultured in EC conditioned media. RESULTS: Compared to ECs cultured in naïve media, ECs exposed to AFC conditioned media expressed higher mRNA and protein levels of key biomarkers of invasive EC phenotype, MMP-2 (2×), MMP-13 (4×), and PDGF-B (1.5-2×), and NGF (24.9 ± 15.2 pg/mL vs 0 in naïve media). Treatment of AF cells with EC culture conditioned media decreased collagen type II expression two fold. Considerable quantities of pro-angiogenic factors IL-8 (396.7 ± 302.0 pg/mL) and VEGF (756.2 ± 375.9 pg/mL) were also detected in the conditioned media of untreated AF cell culture. DISCUSSION: AFCs from degenerated discs secreted factors which stimulated EC production of factors known to induce matrix degradation, angiogenesis, and innervation. IL-8 and VEGF maybe the secreted factors from AFCs which mediate a pro-angiogenic stimulus often implicated in the development of disc degeneration.

Additional malignant breast lesions detected on second-look US after breast MRI vs. additional malignant lesions detected on initial US in breast cancer patients: comparison of US characteristics.

PURPOSE: The purpose of our study was to review and compare the US findings of synchronous malignant breast lesions other than the index cancer additionally detected on second-look US with those detected on initial US, and therefore to determine differing characteristics that may aid in diagnosis and essentially improve the performance of the initial US examination. MATERIALS AND METHODS: A retrospective review of 39 mammographically occult synchronous malignant lesions other than the index cancer from 38 patients was performed (21 lesions: detected on second-look US, 18 lesions: detected on initial US). All patients underwent initial mammography, bilateral whole breast US (BWBU) and breast MRI, and all lesions were confirmed pathologically by biopsy or preoperative localization. RESULTS: Additional malignant breast lesions detected on both initial US and second-look US tended to be subtle and often did not show classic malignant findings. Second-look US lesions (median, 7.0 mm; range, 3 - 22 mm) tended to be smaller than initial US lesions (median, 9.0 mm; range 3 - 45 mm), although the difference was not statistically significant (p = 0.134). Second-look US lesions also showed no posterior acoustic features (p = 0.037) and a significantly higher proportion of lesions with circumscribed or indistinct margins compared to initial US lesions (p = 0.042). Second-look US lesions were significantly subareolar or relatively far (> 5 cm) from the nipple than initial US lesions (p = 0.048). CONCLUSION: Second-look US lesions showed more subtle findings of posterior acoustic features and margins, and tended to be subareolar or relatively far from the nipple compared to initial US lesions. However, both groups showed subtle US findings and there was no significant difference in other features.

A risk-adapted approach using US features and FNA results in the management of thyroid incidentalomas identified by 18F-FDG PET.

PURPOSE: To assess the risk of malignancy of thyroid incidentalomas found on 18F-FDG PET/CT by US features and cytologic results, and to evaluate the clinical usage of a combination of US features and cytology for post-FNA management of thyroid incidentalomas on 18F-FDG PET/CT. MATERIALS AND METHODS: From September 2006 to December 2008, 132 patients with 134 thyroid incidentalomas detected on 18F-FDG PET/CT who had undergone US and US-FNA were included in this study. We evaluated the malignancy rate of thyroid incidentalomas in different subgroups subdivided by US features and US-FNA cytology results. Several variables were compared between the benign and malignant group. RESULTS: The risk of malignancy was 58.2 % (78/132) in thyroid incidentalomas on 18F-FDG PET/CT. Age, gender, and tumor size were not significantly different between the malignant and benign group.  Malignancy rate of thyroid incidentalomas was significantly higher in the suspicious malignant (88.9 %) than in the probably benign group (11.3 %) on US (p < 0.001). Malignancy rates were high in thyroid nodules with "malignancy", "suspicious for malignancy", or "follicular neoplasm" on cytologic results, regardless of US features. However, malignancy rates of thyroid incidentalomas with "unsatisfactory" or "benign" results on cytology were higher in the suspicious malignant (75 %, 12.5 %, respectively) than in the probably benign (0 %) group on US.  CONCLUSIONS: This study demonstrated that the risk of malignancy was high in thyroid incidentalomas on 18F-FDG PET/CT even without suspicious US features. However, there was no malignancy in nodules with no suspicious US features and benign cytology. Based on these results, we concluded that US may not replace FNA in the diagnosis of PET incidentalomas, and that a follow-up may be considered of thyroid incidentalomas with benign cytology and no suspicious US features.

Discordant elastography images of breast lesions: how various factors lead to discordant findings.

PURPOSE: To evaluate the rate of the elastography-pathology discordance, and evaluate which various factors have an effect on discordant elastography images (DEI) of breast lesions. MATERIALS AND METHODS: Elastography images of 284 pathologically confirmed breast lesions of 233 patients were evaluated. Elasticity scores were compared to pathology results, and lesions were divided into 4 groups: benign concordant/discordant, and malignant concordant/discordant. The rate of DEI among benign and malignant lesions was calculated and compared. Patient, lesion factors and image adequacy were compared among the concordant and discordant groups for analysis. RESULTS: Among the 284 breast lesions, 225 (79.2%) were benign, and 59 (20.8%) were malignant. The rate of DEI among malignant lesions was significantly higher than in benign lesions, i. e., 52.5 vs. 3.1% (p < 0.001). Discordant images were more significantly seen in patients with extremely dense breasts on mammography in benign lesions, 42.9 vs. 11.9% (p = 0.034). Discordant images were more significantly seen in malignant lesions < 10 mm or ≥ 20 mm (p = 0.006), and those with inadequate images (64.5 vs. 35.5%, p < 0.001). CONCLUSION: The rate of DEI was higher in malignant lesions than in benign lesions. Dense breast parenchyma, lesion size and image adequacy showed significance in discordant images of elastography which need consideration in image acquisition and interpretation.

False negative results in axillary lymph nodes by ultrasonography and ultrasonography-guided fine-needle aspiration in patients with invasive ductal carcinoma.

PURPOSE: For preoperative evaluation of ALN status using various methods, axillary US and subsequent US-FNA targeting the LNs suspicious for metastasis are the most widely used methods. The purpose of our study was to assess the rate of false-negative results at preoperative ultrasonography (US) and ultrasonography guided fine needle aspiration (US-FNA) of axillary lymph nodes (ALNs) in breast cancer patients and the number of false-negative lymph nodes, and to evaluate factors related to ALN false negative results in US and/or US-FNA in patients diagnosed with invasive ductal carcinoma. MATERIALS AND METHODS: Among 317 patients who underwent surgery for invasive ductal carcinoma during 2009 in Severance hospital, 237 patients had no reported ALN metastasis on preoperative US-FNA and US. We retrospectively reviewed the subsequent surgical pathology and clinicopathologic findings and assessed the rate of false-negative results from US and US-FNA of ALN and the number of false-negative lymph node. We performed univariate analysis and multivariate logistic regression analysis to evaluate the relationships between variable clinicopathologic factors (T-stage, position of ALN, hormone receptors, histologic grade, lymphovascular invasion (LVI) and performance of FNA) and cytologic results (false-negative result; FNALN and true negative result; TNALN) from US and/or US-FNA of ALN. RESULTS: The rate of false-negative results was 42.4 % (59/139) in both US and US-FNA of ALN but among them, 57.6 % (34/59) showed only one metastatic ALN. Breast cancer with FNALN on US and US-FNA was significantly related to positive estrogen receptor (p = 0.003), positive progesterone receptor (p = 0.001), and the presence of LVI (p = 0.004) in univariate analysis. In multivariate analysis, high T stages (≥ T2, odds ratio (OR) 4.007, p = 0.004) and LVI (OR 7.951, p = 0.001) showed significant correlation with FNALN on US and US-FNA. CONCLUSION: More than half of patients with FNALN showed only one metastatic ALN. LVI and high T-stages were the most important factors attributed to FNALN on US and US-FNA in patients with invasive ductal carcinoma.

Is US-guided core needle biopsy (CNB) enough in probably benign nodules with interval growth?

PURPOSE: To investigate whether ultrasound-guided 14-gauge core needle biopsy (US-CNB) is efficient in the diagnosis of probably benign lesions showing interval growth on follow-up US.  MATERIALS AND METHODS: From March 2008 to August 2009, 116 breast lesions in 113 women (mean age: 41.2 years, range: 19 - 63 years) which were initially assessed as category 3 showing interval growth on follow-up US underwent US-CNB, and subsequent US-guided vacuum-assisted excision, surgical excision or follow-up US for at least 12 months. Diagnostic performances of US-CNB were evaluated with histopathologic results and follow-up US as standard reference. Clinical features of the patient and lesions characteristics including follow-up interval (I), the most increased diameter (D), D per I, increased volume (%V) and %V per I were calculated and compared. RESULTS: Of the 116 lesions, 4 lesions were diagnosed as malignancy and 112 as benign on final pathology. Malignancy rate of probably benign lesions showing interval growth was 3.4 % (4/116). Incorrect biopsy rate was 0.9 % (1/116). Palpability or newly developed suspicious US features were more associated with malignancy, 75.0 to 13.4 % and 50.0 to 25.9 %, respectively, but without significance (p = 0.063 and 0.290). Significant differences were seen in average rank when comparing between benign and malignancy in D, %V, D per follow-up interval (I), and %V/I (p = 0.037, 0.017, 0.043 and 0.009, respectively). CONCLUSION: US-CNB is an efficient diagnostic method for probably benign lesions showing interval growth, with discordant biopsy rate of 0.9 %.

Interobserver variability and diagnostic performance in US assessment of thyroid nodule according to size.

PURPOSE: To evaluate the interobserver variability for US assessments of thyroid nodules and analyze the diagnostic performances of US assessments in thyroid nodules according to nodule size. MATERIALS AND METHODS: This was an IRB-approved retrospective study with waiver of informed consent. A total of 400 surgically-confirmed thyroid nodules were included. Nodules were divided into 4 groups by size; group 1 (nodule size < 5 mm), group 2 (5 mm ≤ nodule size < 10 mm), group 3 (10 mm ≤ nodule size < 20 mm), and group 4 (nodule size ≥ 20 mm). Three experienced (7 - 10 years) radiologists retrospectively reviewed the US images. Agreement of each US descriptor and final US assessment, and diagnostic performances were calculated in each group and compared. RESULTS: Composition represented substantial or good agreement (k = 0.719 - 0.89). Margin showed the lowest agreement (k = 0.322 - 0.365). Individual kappa values for final assessment according to nodule size were as follows: group 1 (k = 0.674), group 2 (k = 0.596), group 3 (k = 0.674), and group 4 (k = 0.673). Specificity, PPV, and accuracy were significantly different among the groups with different size (p value < 0.05) and lowest in group 1. NPV, specificity, PPV and accuracy except PPV of observer 3 increased with nodule size (p < 0.05). CONCLUSION: Interobserver agreements were relatively good (k = 0.637) in final US assessment regardless of nodule size in experienced radiologists. High false-positive rate was observed in US assessment in nodules less than 5 mm in maximum diameter.

Interspecies transmission of the canine influenza H3N2 virus to domestic cats in South Korea, 2010.

In the past 4 years, incidences of endemic or epidemic respiratory diseases associated with canine influenza H3N2 virus in Asian dogs have been reported in countries such as South Korea and China. Canine species were considered to be the new natural hosts for this virus. However, at the beginning of 2010, influenza-like respiratory signs, such as dyspnoea, were also observed among cats as well as in dogs in an animal shelter located in Seoul, South Korea. The affected cats showed 100 % morbidity and 40 % mortality. We were able to isolate a virus from a lung specimen of a dead cat, which had suffered from the respiratory disease, in embryonated-chicken eggs. The eight viral genes isolated were almost identical to those of the canine influenza H3N2 virus, suggesting interspecies transmission of canine influenza H3N2 virus to the cat. Moreover, three domestic cats infected with intranasal canine/Korea/GCVP01/07 (H3N2) all showed elevated rectal temperatures, nasal virus shedding and severe pulmonary lesions, such as suppurative bronchopneumonia. Our study shows, for the first time, that cats are susceptible to canine influenza H3N2 infection, suggesting that cats may play an intermediate host role in transmitting the H3N2 virus among feline and canine species, which could lead to the endemic establishment of the virus in companion animals. Such a scenario raises a public health concern, as the possibility of the emergence of new recombinant feline or canine influenza viruses in companion animals with the potential to act as a zoonotic infection cannot be excluded.

The diagnosis of non-malignant papillary lesions of the breast: comparison of ultrasound-guided automated gun biopsy and vacuum-assisted removal.

AIM: To compare the histological upgrade rate of ultrasound (US)-guided vacuum-assisted removal (VAR) and US-14 G-automated core needle biopsy (ACNB) in the diagnosis of papillary breast lesions. MATERIALS AND METHODS: Two hundred and seventy-one biopsies of 230 papillary lesions were examined, which underwent subsequent surgical excision or long-term follow-up after US-ACNB (n = 206) or US-VAR (n = 65). The false-negative and atypical papilloma underestimation rate were compared between the ACNB and VAR groups. Patient and lesion characteristics were collected. The histological upgrade rates of the diagnosis were estimated and compared. RESULTS: Out of 271 papillary lesions, 195 (80.0%) were benign, 21 (7.7%) were atypical, and 55 (20.3%) were malignant. There were no false negatives or underestimated atypical papillomas in the VAR group. However, in the ACNB group, the false-negative rate was 7.6% (12 of 157 benign papillomas, 95% CI; 4.4-12.9%, p = 0.039) and the atypical papilloma underestimation rate was 33% (five of 15 atypical papillomas, 95% CI; 15.2-58.3%, p = 0.135). The histological upgrade rates of the diagnosis for papillary breast lesions were 0% for the VAR (0 of 66) group and 10.2% for the ACNB (21 of 206) group before adjusting for the population (p = 0.003). CONCLUSIONS: ACNB was associated with significantly higher false-negative and histological upgrade rates of diagnosis for papillary breast lesions than VAR.

Value of specimen radiographs in diagnosing multifocality of thyroid cancer.

BACKGROUND: Specimen radiography has been used widely to evaluate the complete excision of calcified breast lesions but has not been evaluated for thyroid cancer. METHODS: Specimen radiographs were evaluated retrospectively to identify additional cancers that were demonstrated only as calcifications. Receiver operating characteristic curve analysis was performed to compare the combination of specimen radiography and ultrasonography versus ultrasonography alone for detecting multifocality. RESULTS: Some 122 thyroid cancer specimens were obtained from 122 patients between January and April 2008. Specimen radiography detected 27 cancers (18.5 per cent) not detected by ultrasonography. Diagnoses were changed after evaluation of specimen radiographs in three of these patients. The area under the curve of the combination of specimen radiography and ultrasonography was significantly higher than that of ultrasonography alone (P = 0.005). CONCLUSION: Specimen radiography is a potentially useful tool for diagnosing cancer type and predicting the extent of thyroid cancer.

Providing essential clinical care for non-COVID-19 patients in a Seoul metropolitan acute care hospital amidst ongoing treatment of COVID-19 patients.

We assessed infection control efforts by comparing data collected over 20 weeks during a pandemic under a dual-track healthcare system. A decline in non-COVID-19 patients visiting the emergency department by 37.6% (P<0.01) was observed since admitting COVID-19 cases. However, patients with acute myocardial infarction (AMI), stroke, severe trauma and acute appendicitis presenting for emergency care did not decrease. Door-to-balloon time (34.3 (± 11.3) min vs 22.7 (± 8.3) min) for AMI improved significantly (P<0.01) while door-to-needle time (55.7 (± 23.9) min vs 54.0 (± 18.0) min) in stroke management remained steady (P=0.80). Simultaneously, time-sensitive care involving other clinical services, including patients requiring chemotherapy, radiation therapy and haemodialysis did not change.

Elimination of porcine reproductive and respiratory syndrome virus from a seedstock breeding farm and a supplying boar stud by a modified test and removal method.

To eliminate porcine reproductive and respiratory syndrome virus (PRRSV) from a supplying boar stud, samples of serum and semen from 118 boars were assessed three times a month by an indirect fluorescent antibody (IFA) test to detect antibodies, and by a nested reverse transcriptase-PCR (nRT-PCR) to detect the genome of PRRSV. The boars detected as persistently infected carriers were culled. A PRRSV-negative population of boars was established after three months and no semen positive for the virus was detected for six months. Subsequently, a three-step plan was introduced to eliminate PRRSV from the seedstock breeding farm during three parity cycles on the farm over 15 months, each step taking five months. In step 1, umbilical cords taken from piglets at birth and serum samples taken from their dams at the start of weaning were subjected to ifa and nRT-pcr analysis. The sows with PRRSV detected in serum by nRT-pcr were regarded as carrier sows and culled. The rates of detection of PRRSV were reduced from 5 percent to 2.5 percent in the sera of the sows, and from 14.8 percent to 1.8 percent in the umbilical cords of the piglets. In step 2, the sows that had farrowed the piglets with PRRSV detected by nRT-PCR in their cords were considered to have transmitted the infection and removed. During step 2, the virus detection rates in umbilical cords by nRT-pcr were reduced, but not completely eliminated. In step 3, 10-week-old nursery pigs with antibodies to PRRSV in their serum by ifa and elisa were culled. The three steps established the PRRSV-negative state of the multisite farm containing the breeding and nursery farm, and the PRRSV-negative state of both the multisite farm and the supplying boar stud was evaluated by monthly monitoring over at least one parity cycle of the farm for five months.

Merlin facilitates ubiquitination and degradation of transactivation-responsive RNA-binding protein.

The Nf2 tumor suppressor codes for merlin, a protein whose function is largely unknown. We have previously demonstrated a novel interaction between merlin and TRBP, which inhibits the oncogenic activity of TRBP. In spite of the significance of their functional interaction, its molecular mechanism still remains to be elucidated. In this report, we investigated how merlin inhibits the oncogenic activity of TRBP in association with cell growth conditions. In the human embryonic kidney 293 cell line, the level of endogenous merlin increased, whereas that of endogenous TRBP significantly decreased along with the increase in cell confluence. We demonstrated that the carboxyl-terminal region of TRBP was responsible for this phenomenon using stable cell lines expressing deletion mutants of TRBP. The overexpression of merlin decreased the protein level of TRBP, and the ubiquitin-like subdomain of merlin's FERM domain was important for this activity. We also demonstrated that TRBP is ubiquitinylated and the ubiquitinylated forms of TRBP are accumulated by ectopically expressed merlin or cell confluence in the presence of MG132, a proteasome inhibitor. Furthermore, we showed that the regulation of TRBP in response to cell confluence was abolished upon knockdown of merlin expression by specific small interfering RNA. Finally, we showed that ectopically expressed merlin restored cell-cell contact inhibition in cells stably expressing TRBP but not in TRBPDeltac. These results suggest that merlin is involved in the regulation of TRBP protein level by facilitating its ubiquitination in response to such cues as cell-cell contacts.

Oral efficacy of Vero cell attenuated porcine epidemic diarrhea virus DR13 strain.

A Vero cell attenuated porcine epidemic diarrhea virus (PEDV) strain, DR13, was distinguished from wild-type PEDV using restriction enzyme fragment length polymorphism (RFLP). Cell attenuated DR13 was orally or intramuscularly (IM) administered to late-term pregnant sows, and mortality resulting from the highly virulent PEDV challenge was investigated in passively immunized suckling piglets of the two different groups. The mortality rate of the oral group (13%) was lower than that of the IM group (60%). In particular, the concentration of IgA against PEDV was higher in piglets of sows in the oral group, compared to the IM group. The attenuated DR13 virus remained safe, even after three backpassages in piglets. The findings of this study support the theory that the Vero cell attenuated DR13 virus may be applied as an oral vaccine for inducing specific immunity in late-term pregnant sows with a high margin of protection against PEDV infection.

Inhibition of Porcine Endogenous Retrovirus by Multi-Targeting Micro RNA Against Long Terminal Region.

BACKGROUND: There might be much benefit in xenotransplantation, however, the risk of infections across species barriers remains, especially porcine endogenous retrovirus (PERV). To date, many attempts have been made to knock down active PERVs by inhibitory RNA (RNAi) and micro RNA (miRNA), which target different genes of PERV. There are a few studies that have explored whether targeting promoter regions of PERV could exert an inhibition effect. METHODS: miRNAs were automatically selected based on an online program BLOCK-iT RNAi Designer. The inhibition efficiency between miRNAs was compared based on their inhibition of different PERV genes: long terminal repeats (LTR), gag, and pol. Both relative quantitative real-time polymerase chain reaction (PCR) and C-type reverse transcriptase activity were performed. RESULTS: The results demonstrated that miRNA targeting the LTR region degraded the target sequence, and simultaneously inhibited the mRNA expression of both gag and pol genes of PERV. The LTR1, LTR2, and dual LTR1 + LTR2 miRNA inhibited 76.2%, 22%, and 76.8% of gag gene expression, respectively. Similarly, the miRNA was found to knock down the pol gene expression of 69.8%, 25.5%, and 77.7% for single targeting miRNA (LTR1 and LTR2) and multi-targeting miRNA (LTR1 + LTR2), respectively. A stable PK15 clone constitutively expressed dual LTR1 + LTR2 miRNA and exhibited higher inhibitory up to 82.8% and 92.7% of the expressions of the gag and pol genes, respectively. Also, the result of co-cultivation of dual LTR1 + LTR2 miRNA transfected PK15 cell with a human cell line inhibited expression of LTR, gag, and pol genes of PERV. CONCLUSIONS: In conclusion, this study suggested that the LTR might be an alternative target for gene silencing of PERV, and that multi-targeting miRNA had better inhibitory effect than single- targeting miRNA. In an in vitro model, the presence of miRNA was able to reduce PERV infectivity in a human cell line.