[Modified gasless trans-subclavian approach endoscopic lateral neck dissection for treatment of papillary thyroid carcinoma: a series of 31 cases].

Objective: To examine the feasibility of the modified gasless trans-subclavian approach endoscopic thyroidectomy for lateral neck dissection (LND) in papillary thyroid carcinoma (PTC). Methods: The clinical data of 31 patients with PTC who underwent modified gasless trans-subclavian approach endoscopic LND in the Department of Head and Neck Surgery, Run Run Shaw Hospital, from January to October 2022 were retrospectively analyzed. There were 2 males and 29 females, aged (32.6±8.3) years (range: 17 to 55 years). The maximum diameter of the primary thyroid lesion (M(IQR)) was 1.06 (1.16) cm (range: 0.53 to 2.44 cm), and the maximum diameter of the metastatic lymph node was (1.04±0.37) cm (range: 0.44 to 1.88 cm). Operation time, postoperative hospital stay, number of lymph nodes dissected, and postoperative complications were recorded. Outpatient follow-up was conducted until November 30, 2022. Results: All operations were successfully completed with the endoscopy approach without conversion to open surgery. The operation time was 160 (20) minutes (range: 100 to 215 minutes), and the postoperative hospital stay was 4 (2) days (range: 2 to 14 days). The number of lymph nodes obtained by dissection in the central and lateral compartment of the neck was 11 (12) (range: 0 to 37) and 34.7±14.8 (range: 15 to 69), respectively. Temporary hypoparathyroidism occurred in 4 cases and all recovered within 1 month after the operation. One case suffered from recurrent laryngeal nerve injury (continuing followed up to assess whether it is a temporary injury). The complication of LND included 1 case of chylous leakage that was recovered with conservative treatment, 1 case of Horner syndrome returned to normal 3 months after surgery. During follow-up, there was no residual tumor or recurrence. Conclusion: The modified gasless trans-subclavian approach endoscopic LND for PTC is feasible, with a thorough dissection and concealed incision.

[Gasless submental-transoral combined appoach endoscopic thyroidectomy for papillary thyroid carcinoma: a series of 41 cases].

Objective: To examine the safety and feasibility of gasless submental-transoral combined appoach endoscopic thyroidectomy for papillary thyroid carcinoma (PTC). Methods: A retrospective analysis of the clinical data of 41 patients with PTC who underwent the gasless submental-transoral combined appoach endoscopic thyroidectomy at the Department of Head and Neck Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine from November 2020 to April 2021. There were 5 males and 36 females with the age of (35.0±8.7) years (range: 19 to 58 years). A horizontal incision with a length of 2.0 cm is made under the chin as an observation hole, a 10 mm Trocar and a self-developed retractor are inserted, and two 5 mm longitudinal incisions are made on the labial side in the vestibule of the oral cavity as an operation hole, each inserting a 5 mm Trocar, the operation direction is from the cranial side to the caudal side. The sensation of the lower lip and chin was measured on the first day and one month postoperative. The operation time, hospital stay, the number of lymph nodes dissected and postoperative complications were recorded. Results: Surgical procedures in all cases were successfully completed under endoscopic approach without transfering to open surgery. The operation time was (99±34) minutes (range: 50 to 180 minutes) and the postoperative hospital stay was (3.4±2.2) days (range: 2 to 16 days). The maximum diameter of PTC was (7.6±5.8) mm (range: 2 to 30 mm), and the number of lymph nodes of the central compartment dissection was 6(5) (M(IQR)) (range: 1 to 25). The duration of follow-up is 1 month after operation, and the follow-up method is adopted in outpatient clinic. Postoperation complications included 2 cases of transient hypoparathyroidism, One case of recurrent laryngeal nerve injury (continue to follow up to assess whether it is a temporary injury). Postoperative minor chyle leak, seroma, and local redness and swelling in 1 case each were cured after conservative treatment. 1 case of transient minor numbness of the lower lip was observed. No permanent hypoparathyroidism, postoperative bleeding and numbness of the chin was observed. Conclusion: The gasless submental-transoral combined appoach endoscopic thyroidectomy is a feasible approach in selected PTC patients and has clinical application value.

Application of virome capture sequencing in shellfish sold at retail level in Singapore.

During the period from late 2019 to early 2020, we performed a foodborne virus detection from shellfish collected in Singapore at retail level. Multiple human enteric viruses were included as our targets including human noroviruses (NoVs) GI and GII, hepatitis A virus, hepatitis E virus and rotavirus. Out of the 60 shellfish samples, 23 (38·3%) were detected to be positive by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) with human enteric viruses. Six samples were selected to proceed with virome capture sequencing with positive control samples spiked with serially diluted NoV GII clinical samples in oyster extract. As a result, the natural sample with comparable Ct values (34·0-35·0) of the spiked sample as detected by RT-qPCR generated much lower read counts (>7-log2 cumulative sum scaling difference) and genome coverage (406 nt. vs 3715 nt.), suggesting that the RT-qPCR positive signals detected from the shellfish samples collected at the retail market were likely from degraded RNA derived from inactive virus particles.

Cost-effectiveness of mifepristone and misoprostol versus misoprostol alone for the management of missed miscarriage: an economic evaluation based on the MifeMiso trial.

OBJECTIVE: To assess the cost-effectiveness of mifepristone and misoprostol (MifeMiso) compared with misoprostol only for the medical management of a missed miscarriage. DESIGN: Within-trial economic evaluation and model-based analysis to set the findings in the context of the wider economic evidence for a range of comparators. Incremental costs and outcomes were calculated using nonparametric bootstrapping and reported using cost-effectiveness acceptability curves. Analyses were performed from the perspective of the UK's National Health Service (NHS). SETTING: Twenty-eight UK NHS early pregnancy units. SAMPLE: A cohort of 711 women aged 16-39 years with ultrasound evidence of a missed miscarriage. METHODS: Treatment with mifepristone and misoprostol or with matched placebo and misoprostol tablets. MAIN OUTCOME MEASURES: Cost per additional successfully managed miscarriage and quality-adjusted life years (QALYs). RESULTS: For the within-trial analysis, MifeMiso intervention resulted in an absolute effect difference of 6.6% (95% CI 0.7-12.5%) per successfully managed miscarriage and a QALYs difference of 0.04% (95% CI -0.01 to 0.1%). The average cost per successfully managed miscarriage was lower in the MifeMiso arm than in the placebo and misoprostol arm, with a cost saving of £182 (95% CI £26-£338). Hence, the MifeMiso intervention dominated the use of misoprostol alone. The model-based analysis showed that the MifeMiso intervention is preferable, compared with expectant management, and this is the current medical management strategy. However, the model-based evidence suggests that the intervention is a less effective but less costly strategy than surgical management. CONCLUSIONS: The within-trial analysis found that based on cost-effectiveness grounds, the MifeMiso intervention is likely to be recommended by decision makers for the medical management of women presenting with a missed miscarriage. TWEETABLE ABSTRACT: The combination of mifepristone and misoprostol is more effective and less costly than misoprostol alone for the management of missed miscarriages.

Fallopian tube catheterization in the treatment of proximal tubal obstruction: a systematic review and meta-analysis.

Study question: What is the chance of clinical pregnancy when fallopian tube catheterization is used for proximal tubal obstruction? Summary answer: The pooled clinical pregnancy rate of tubal catheterization after proximal tubal obstruction is 27% (95% CI 25-30%). What is known already: Restoring fallopian tube patency by performing tubal catheterization has fallen out of favour since the increased availability of IVF. Our study is the first systematic review and meta-analysis to investigate reproductive outcomes following tubal catheterization for proximal tubal obstruction. Study design, size, duration: We undertook a systematic review and meta-analysis of 27 observational studies consisting of 1720 patients undergoing tubal catheterization for proximal tubal obstruction, who attempted to conceive naturally after the procedure. Participants/materials, setting, methods: Systematic literature searches were performed in MEDLINE, EMBASE and the Cochrane Central Register of Controlled Trials. A total of 2195 titles and abstracts were reviewed. Only studies that reported outcomes when tubal catheterization was performed with no other tubal surgery were included. Twenty-seven cohort studies matched the inclusion criteria for the meta-analysis. Main results and the role of chance: The meta-analysis showed a pooled clinical pregnancy rate of 27% (95% CI 25-30%) after the use of tubal catheterization for unilateral or bilateral proximal tubal obstruction (27 studies, 1556 patients). In women with bilateral obstruction (14 studies, 617 patients), the clinical pregnancy rate was 27% (95% CI 23-32%). Our meta-analysis demonstrated that the pooled cumulative clinical pregnancy rates were 22.3% (95% CI 17.8-27.8%) at 6 months, 25.8% (95% CI 21.1-31.5%) at 9 months, 26.4% (95% CI 23.0-30.2%) at 12 months, 26.0% (95% CI 22.8-29.7%) at 18 months, 27.0% (95% CI 24.0-30.5%) at 24 months, 27.9% (95% CI 24.9-31.3%) at 36 months and 28.5% (95% CI 25.5-31.8%) at 48 months. The pooled live birth rate (14 studies, 551 patients) was 22% (95% CI 18-26%). The pooled ectopic pregnancy rate (27 studies, 1556 patients) was 4% (95% CI 3-5%). The included studies scored satisfactorily on the Newcastle-Ottawa quality assessment scale. Limitations, reasons for caution: The pooled clinical pregnancy rate after tubal catheterization was found to be almost comparable to that after IVF. However, included studies were small, non-comparative series with significant clinical heterogeneity in population characteristics, follow-up and surgical equipment, technique and experience. Wider implications of the findings: These findings suggest fallopian tube catheterization as an alternative strategy to IVF in patients presenting with proximal tubal obstruction. Further research should focus on comparing different surgical techniques of fallopian tube catheterization with IVF and provide cumulative reproductive outcomes over long-term follow-up. Study funding/competing interest(s): No funding was required and the authors have no competing interests to declare. Registration number: N/A.

Inhibition of West Nile virus replication in cells stably transfected with vector-based shRNA expression system.

In this study, the efficacies of short hairpin RNAs (shRNAs) targeting different regions of West Nile virus (WNV) strain Sarafend genome were investigated. Short hairpin RNAs targeting Capsid, NS2B and NS4B genes were cloned into pSilencer 3.1-H1 neo and designated as pshCapsid, pshNS2B and pshNS4B, respectively. Vero cells that were positively transfected were selected for creating stable cell lines expressing shRNAs constitutively. These cells were subjected to West Nile virus at multiplicity of infection (M.O.I.) of 10. The cells stably transfected with pshCapsid gave the best silencing effect among the three stable cell lines (transfected with pshCapsid, pshNS2B and pshNS4B) at both 12- and 24 h p.i. When compared to the non-transfected WNV-infected cells, pshCapsid stably transfected cells showed more than 4 log(10) unit reduction in viral transcripts and greater than 3 log(10) unit reduction in virus production. Cells stably transfected with pshNS2B did not exhibit as strong an inhibition when compared to the pshCapsid stably transfected cells having only 2 log(10) unit reduction in virus titre. The pshNS4B-stably transfected cells did not suppress WNV replication. Hence, from this study, pshCapsid has the potential to be developed into effective antiviral strategy for WNV infection.

TNF receptor I polymorphism is associated with persistent palindromic rheumatism.

OBJECTIVES: To investigate the association between tumour necrosis factor-alpha (TNFalpha), TNF receptor superfamily member 1A (TNFRSF1A, also known as TNFRI), TNFRSF1B (TNFRII), and interleukin-1beta (IL-1beta) single nucleotide polymorphisms (SNPs) and the susceptibility to persistent palindromic rheumatism (PR). METHODS: Fifty-six unrelated patients with persistent PR and 100 unrelated healthy controls were genotyped for TNFalpha -308G/A, -238G/A, and +488G/A, TNFRSF1A -609G/T and +36A/G, TNFRSF1B +676T/G and +1663G/A, and IL-1beta -511C/T, -31T/C, and +3954C/T using real-time polymerase chain reaction (RT-PCR). RESULTS: The TNFRSF1A +36G allele [odds ratio (OR) = 3.94, p = 0.003, corrected p (p(c)) = 0.03] and the TNFRSF1A +36AG genotype (OR = 4.81, p = 0.002, p(c) = 0.04) were significantly associated with persistent PR. The frequency of TNFRSF1B +676T/+1663A was increased in PR patients (OR = 2.12, p = 0.01), but failed to reach statistical significance after Bonferroni correction. No correlation was observed between persistent PR and TNFalpha, TNFRSF1A -609G/T, or IL-1beta SNPs. CONCLUSIONS: The results of this study provide evidence of an association between persistent PR and SNPs within the TNFRSF1A gene, and suggest that TNFRI is involved in the aetiopathogenesis of PR.

c-Src protein kinase inhibitors block assembly and maturation of dengue virus.

Dengue virus is a mosquito-borne flavivirus that represents an important emerging infectious disease and is an international health concern. Currently, there is no vaccine or effective antiviral therapy to prevent or to treat dengue virus infection. The slow progress in developing antiviral agents might be alleviated by the availability of efficient high-throughput anti-dengue virus screening assays. In this study, we report an immunofluorescence image-based assay suitable for identification of small molecule inhibitors of dengue virus infection and replication. Using this assay, we have discovered that inhibitors of the c-Src protein kinase exhibit a potent inhibitory effect on dengue virus (serotypes 1-4) and murine flavivirus Modoc. Mechanism of action studies demonstrated that the c-Src protein kinase inhibitor dasatinib prevents the assembly of dengue virions within the virus-induced membranous replication complex. These results demonstrate that this cell-based screen may provide a powerful means to identify new potential targets for anti-dengue drug development while simultaneously providing pharmacological probes to investigate dengue virus-host cell interactions at the biochemical level. Given the simplicity and excellent reproducibility of the assay, it should be useful in high-throughput screens of both small molecule and RNAi libraries when implemented on a robotic image-based high-throughput screen (HTS) platform. Given the reasonable clinical safety of inhibitors such as dasatinib and AZD0530, inhibitors of c-Src protein kinase may have the potential to become a new class of anti-dengue viral therapeutic agents.

The envelope glycoprotein domain III of dengue virus serotypes 1 and 2 inhibit virus entry.

Dengue virus (DV) is a flavivirus and its urban transmission is maintained largely by its mosquito vectors and vertebrate host, often human. In this study, investigation was carried out on the involvement of domain III of the envelope (E) glycosylated protein of dengue virus serotypes 1 and 2 (DV-1 and DV-2 DIII) in binding to host cell surfaces, thus mediating virus entry. Domain III protein of flavivirus can also serve as an attractive target in inhibiting virus entry. The respective DV DIII proteins were expressed as soluble recombinant fusion proteins before purification through enzymatic cleavage and affinity purification. The purified recombinant DV-1 and DV-2 DIII proteins both demonstrated the ability to inhibit the entry of DV-1 and DV-2 into HepG2 cells and C6/36 mosquito cells. As such, the DV DIII protein is indeed important for the interaction with cellular receptors in both human and mosquito cells. In addition, this protein induced antibodies that completely neutralized homologous dengue serotypes although not with the same efficiency among the heterologous serotypes. This observation may be of importance when formulating a generic vaccine that is effective against all dengue virus serotypes.

Versatile use of extra-corporeal life support to resuscitate acute respiratory distress patients.

Extra-corporeal life support (ECLS) has been applied successfully to congenital respiratory defects but less optimally to acquired pulmonary failure. We extended this support to certain extreme complexities of patients with acute respiratory distress. From January 2003 to June 2005, 16 (nine men and seven women) patients refractory to ventilator support were treated with ECLS. Their median age was 32.4 years (1.5-70). The triggering events were pulmonary haemorrhage (n = 4), pneumonia (n = 7), aspiration (n = 2) and pancreatitis (n = 3). The indications for support were hypoxaemia in 13 and hypercapnia in three patients. Ten (63%) met the criteria of fast entry. Thirteen (81%) received veno-venous (V-V) mode support and the other three received veno-arterial mode support initially, but then converted to V-V mode after sufficient oxygenation stabilised haemodynamics. Initial pump flow was maximised to improve (mean 3250 +/- 1615 ml/min) to improve the oxygenation. Four patients with active pulmonary haemorrhage were heparin free in the first 12-24 h of support without complications. Excluding one prematurely terminated patient because of brain permanent damage, the duration of support was 162 +/- 95 h (67-363). Eleven (69%) weaned successfully from ECLS and 10 (63%) discharged and regained normal pulmonary performance in a median of 26.8 months follow-up. Pulmonary support using ECLS was feasible in selected patients with acute respiratory distress. Modification of guidelines for liberal use, early deployment before secondary organ damage and prevention of complications during support were the key to final success.

Expression of vector-based small interfering RNA against West Nile virus effectively inhibits virus replication.

RNA interference is one of the effective emerging anti-viral strategies to inhibit virus infection in cells. In this study, a small interfering RNA expressing vector (pSilencer-NS5) targeting the NS5 gene of West Nile virus (WNV) was employed to target and destroy WNV transcripts. Real-time PCR revealed drastic reduction in WNV RNA transcripts in pSilencer-NS5-transfected Vero cells. The virus infectious titre was also significantly reduced by 90% as determined by plaque assays. The resulting decrease in virus replication was shown to be specific since both scrambled and nucleotide(s) mismatch siRNA against WNV NS5 gene did not have any effect on WNV productive yields. Furthermore, Western immunoblot analysis on the expression of viral NS5 and envelope (E) proteins showed significant down-regulation on the expression of viral NS5 and envelope (E) proteins in virus-infected cells that were pre-transfected with pSilencer-NS5. These data clearly supported the notion that the expression of vector-based siRNA against WNV NS5 gene is able to exert its silencing effect on WNV-infected cells without inducing cytotoxicity, hence holding promise in therapeutic treatment of this important emerging infectious disease.

Analysis of the endocytic pathway mediating the infectious entry of mosquito-borne flavivirus West Nile into Aedes albopictus mosquito (C6/36) cells.

The initial interaction between mosquito-borne flavivirus West Nile and mosquito cells is poorly characterized. This study analyzed the endocytic and the associated signaling pathway that mediate the infectious entry of West Nile virus (WNV) into mosquito cell line (C6/36). Pretreatment of C6/36 cells with pharmacological drugs that blocks clathrin-mediated endocytosis significantly inhibited virus entry. Furthermore, the transfection of functional blocking antibody against clathrin molecules and the overexpression of dominant-negative mutants of Eps15 in C6/36 cells caused a marked reduction in WNV internalization. WNV was shown to activate focal adhesion kinase (FAK) to facilitate the endocytosis of virus but not the mitogen-activated protein kinases (ERK1 and ERK2). Subsequent to the internalization of WNV, the virus particles are translocated along the endosomal pathway as revealed by double-immunofluorescence assays with anti-WNV envelope protein and cellular markers for early and late endosomes. Specific inhibitor for protein kinase C (PKC) was shown to be highly effective in blocking WNV entry by inhibiting endosomal sorting event. The disruption of the microtubule network using nocodazole also drastically affects the entry process of WNV but not the disruption of actin filaments by cytochalasin D. Finally, a low-pH-dependent step is required for WNV infection as revealed by the resistance of C6/36 cells to WNV infection in the presence of lysosomotropic agents.

Characterization of plasma membrane-associated proteins from Aedes albopictus mosquito (C6/36) cells that mediate West Nile virus binding and infection.

This study isolated and characterized the West Nile virus (WNV) putative receptor molecule(s) from Aedes albopictus mosquito (C6/36) cells. The binding of WNV to C6/36 cells was saturated with 5000 particles per cell. The entry of WNV into C6/36 cells was strongly inhibited when pretreated with proteinase K and to a lesser extent with sodium periodate. However, pretreatment of C6/36 cells with phospholipases, glycosidases, heparinases and neurimidase had no effect on virus entry. By using virus overlay protein blot assay, WNV was observed to bind to the 140-kDa, 95-kDa, 70-kDa and 55-kDa plasma membrane-associated molecules isolated from C6/36 cells. Murine antibodies generated against the 95-kDa and 70-kDa membrane proteins effectively blocked WNV, Japanese encephalitis virus (JEV) and Dengue virus (DV) serotype 2 infection in C6/36 cells. In addition, the binding of the recombinant-WNV envelope domain III protein to C6/36 cells can be inhibited by the anti-95-kDa and anti-70-kDa membrane protein antibodies. These data strongly supported the possibility that the 95-kDa and 70-kDa plasma membrane-associated proteins are part of a receptor complex for mosquito-borne flaviviruses (WNV, JEV and DV) on mosquito cells.

Mycotic aneurysm of the superior mesenteric artery in a young woman.

Aneurysm of the superior mesenteric artery (SMA) is rare. We, in this study, present the case of a 21-year-old woman with a history of heroin abuse who was admitted to our hospital for infective endocarditis complicated by floating vegetation at the posterior mitral valve. After receiving 2-week antibiotic treatment, the patient had acute abdominal pain. Computed tomography demonstrated an aneurysm at the SMA. The mycotic aneurysm was resected and the mitral valve was repaired successfully. This report reviews the pathophysiology of mycotic aneurysms of the SMA and role of computed tomography in the differential diagnosis of this condition from acute mesenteric ischaemia.

Inhibition of West Nile virus entry by using a recombinant domain III from the envelope glycoprotein.

The envelope glycoprotein located at the outermost surface of the flavivirus particle mediates entry of virus into host cells. In this study, the involvement of domain III of West Nile virus (WNV-DIII) envelope protein in binding to host cell surface was investigated. WNV-DIII was first expressed as a recombinant protein and purified after a solubilization and refolding procedure. The refolded WNV-DIII protein displays a content of beta-sheets consistent with known homologous structures of other flavivirus envelope DIII, shown by using circular dichroism analysis. Purified recombinant WNV-DIII protein was able to inhibit WNV entry into Vero cells and C6/36 mosquito cells. Recombinant WNV-DIII only partially blocked the entry of dengue-2 (Den 2) virus into Vero cells. However, entry of Den 2 virus into C6/36 was blocked effectively by recombinant WNV-DIII. Murine polyclonal serum produced against recombinant WNV-DIII protein inhibited infection with WNV and to a much lesser extent with Den 2 virus, as demonstrated by plaque neutralization assays. Together these results provided strong evidence that immunoglobulin-like DIII of WNV envelope protein is responsible for binding to receptor on the surface of host cells. The data also suggest that similar attachment molecule(s) or receptor(s) were used by WNV and Den 2 virus for entry into C6/36 mosquito cells.

Infectious entry of West Nile virus occurs through a clathrin-mediated endocytic pathway.

The pathway of West Nile flavivirus early internalization events was mapped in detail in this study. Overexpression of dominant-negative mutants of Eps15 strongly inhibits West Nile virus (WNV) internalization, and pharmacological drugs that blocks clathrin also caused a marked reduction in virus entry but not caveola-dependent endocytosis inhibitory agent, filipin. Using immunocryoelectron microscopy, WNV particles were seen within clathrin-coated pits after 2 min postinfection. Double-labeling immunofluorescence assays and immunoelectron microscopy performed with anti-WNV envelope or capsid proteins and cellular markers (EEA1 and LAMP1) revealed the trafficking pathway of internalized virus particles from early endosomes to lysosomes and finally the uncoating of the virus particles. Disruption of host cell cytoskeleton (actin filaments and microtubules) with cytochalasin D and nocodazole showed significant reduction in virus infectivity. Actin filaments are shown to be essential during the initial penetration of the virus across the plasma membrane, whereas microtubules are involved in the trafficking of internalized virus from early endosomes to lysosomes for uncoating. Cells treated with lysosomotropic agents were largely resistant to infection, indicating that a low-pH-dependent step is required for WNV infection. In situ hybridization of DNA probes specific for viral RNA demonstrated the trafficking of uncoated viral RNA genomes to the endoplasmic reticulum.

Apoptosis in primary cardiac tumours.

Apoptosis, or programmed cell death, is now recognised as an important cellular event during both normal development and specific disease progression. Apoptosis has been suggested to play a critical role in several cardiovascular diseases, but has not yet been identified as a major influence in primary cardiac tumours. A retrospective review of the achieved material at Chang Gung Memorial Hospital revealed seven patients with cardiac myxoma and one with a tumour originating from the crista terminalis, from January 2002 to December 2002. The medical chart, surgical pathology reports and microscopic slides were available in all cases. All patients, including eight cardiac myxomas and one tumour from crista terminalis, were assessed for apoptosis by terminal deoxynucleotidyl transferase nick-end labelling assay. In this study, apoptosis is well documented in all seven myxoma and has even been reported in tumour from the crista terminalis. Interestingly, apoptosis appears related to the nature of the cell properties rather than the incidence of embolism. In conclusion, apoptosis is important in the progression of the primary cardiac tumours, but the mechanism of cardiac tumour regression still remains uncertain.

Cyclosporine enhances vasorelaxation in coronary but not pulmonary artery after 16-hour preservation with UW solution.

Cyclosporine (CsA), a calcineurin inhibitor, has been associated with endothelial dysfunction in transplant patients. Human and in vitro studies suggest that CsA produces endothelial dysfunction by impairing vascular endothelium-dependent relaxation. However, little is know about the CsA effects to modulate the vasorelaxation after prolonged graft preservation. In this study using a protocol designed to eliminate the influences of infusion pressure and shear stress, we evaluated the effect of CsA on vasorelaxation of coronary and pulmonary arteries after 16-hour University of Wisconsin (UW) solution preservation.

The mechanism of cell death during West Nile virus infection is dependent on initial infectious dose.

The mechanism of West Nile (WN) virus-induced cell death is determined by the initial infectious dose. In Vero cells infected with WN virus at an m.o.i. of 10 or greater, morphological changes characteristic of necrosis were observed as early as 8 h post-infection (p.i.). Pathological changes included extensive cell swelling and loss of plasma membrane integrity, as revealed by optical and electron microscopy. High extracellular lactate dehydrogenase (LDH) activity was observed together with leakage of the high mobility group 1 (HMGB1) protein into the extracellular space. When cells undergo necrosis, they release the HMGB1 protein, a pro-inflammatory mediator cytokine. At high infectious doses, loss of cell plasma membrane integrity was due to the profuse budding of WN progeny virus particles during maturation. When this profuse budding process was disrupted using cytochalasin B, LDH activity was reduced dramatically. In contrast, WN virus-induced cell killing occurred predominantly by apoptosis when cells were infected with an m.o.i. of

Actin filaments participate in West Nile (Sarafend) virus maturation process.

West Nile (Sarafend) virus has previously been shown to egress by budding at the plasma membrane of infected cells, but relatively little is known about the mechanism involved in this mode of release. During the course of this study, it was discovered that actin filaments take part in the virus maturation process. Using dual-labeled immunofluorescence and immunoelectron microscopy at late infection (10 hr p.i.), co-localization of viral structural (envelope and capsid) proteins with actin filaments was confirmed. The virus structural proteins were also immunoprecipitated with anti-actin antibody, further demonstrating the strong association between the two components. Perturbation of actin filaments by cytochalasin B strongly inhibited the release of West Nile virus (approximately 10,000-fold inhibition) when compared with the untreated cells. Infectious virus particles were recovered after the removal of cytochalasin B. Further confirmation was obtained when nucleocapsid particles were found associated with disrupted actin filaments at the periphery of cytochalasin B-treated cells. Together, these results showed that actin filaments do indeed have a key role in the release of West Nile (Sarafend) virions.