[Short-term and long-term survival in sleeve lobectomy by video-assisted thoracic surgery versus thoracotomy basing on the propensity score matching].

Objective: To evaluate the safety and efficacy of patients with centrally located lung cancer in sleeve lobectomy by video-assisted thoracic surgery (VATS). Methods: A retrospective analysis was performed on consecutive patients with centrally located lung cancer who underwent sleeve lobectomy admitted in the Affiliated Hospital of Qingdao University from January 2010 to September 2014. Propensity score matching analysis was performed to compare patients for thoracoscopic surgery and open surgery. Twenty-one pairs (42 cases) patients were included for analysis. The t-test, χ(2) test or Fisher's exact probabilities was adopted, if appropriate, to compare demographics and outcomes between the 2 groups. The Kaplan-Meier method and the Log-rank test were used for the distributions of disease free survival (DFS) and overall survival (OS) and their comparisons. Results: After propensity score-matched analysis, the VATS group had a longer operative time ((296.9±73.6) minutes vs. (218.1±59.2) minutes, t=3.82, P=0.00), but shorter postoperative drainage time ((3.3±1.5) days vs. (2.0±3.0) days, t=-0.93, P=0.01) and hospitalization time((6.7±2.8) days vs. (12.1±8.7)days, t=-1.72, P=0.01) than that of the thoracotomy group. Perioperative complications, 1-year and 3-year disease-free and overall survival rates were not statistically different between the two groups. Conclusion: For suitable patients, sleeve lobectomy by VATS is an acceptable safe and effective surgical procedure for patients with central lung cancer.

The effects of compound stimulus extinction and inhibition of noradrenaline reuptake on the renewal of alcohol seeking.

Alcohol-related stimuli can trigger relapse of alcohol-seeking behaviors even after extended periods of abstinence. Extinction of such stimuli can reduce their impact on relapse; however, the expression of extinction can be disrupted when testing occurs outside the context where extinction learning took place, an effect termed renewal. Behavioral and pharmacological methods have recently been shown to augment extinction learning; yet, it is not known whether the improved expression of extinction following these treatments remains context-dependent. Here we examined whether two methods, compound-stimulus extinction and treatment with the noradrenaline reuptake inhibitor atomoxetine, would reduce the vulnerability of extinction to a change in context. Following alcohol self-administration, responding was extinguished in a distinct context. After initial extinction, further extinction was given to a target stimulus presented in compound with another alcohol-predictive stimulus intended to augment prediction error (Experiment 1) or after a systemic injection of atomoxetine (1.0 mg kg(-1); Experiment 2). A stimulus extinguished as part of a compound elicited less responding than a stimulus receiving equal extinction alone regardless of whether animals were tested in the training or extinction context; however, reliable renewal was not observed in this paradigm. Importantly, atomoxetine enhanced extinction relative to controls even in the presence of a reliable renewal effect. Thus, extinction of alcohol-seeking behavior can be improved by extinguishing multiple alcohol-predictive stimuli or enhancing noradrenaline neurotransmission during extinction training. Importantly, both methods improve extinction even when the context is changed between extinction training and test, and thus could be utilized to enhance the outcome of extinction-based treatments for alcohol-use disorders.

NOVEL LEAD ZIRCONATE TITANATE COMPOSITE VIA FREEZING TECHNOLOGY FOR HIGH FREQUENCY TRANSDUCER APPLICATIONS.

Novel PZT-5A ceramic-polymer composite was prepared via freezing technology. This composite exhibited good dielectric and ferroelectric behaviors. At 1 kHz, the dielectric constant and the dielectric loss were 546 and 0.046, respectively, while the remnant polarization was 13.0 µC/cm(2) at room temperature. The electromechanical coupling coefficient (k(t)) of PZT-5A composite was measured to be 0.54, which is similar to that of PZT piezoelectric ceramic. The piezoelectric coefficient (d(33)) of PZT-5A composite was determined to be ~250 pC/N. Using this composite, a 58MHz single element transducer with the bandwidth of 70% at -6dB was built, and the insertion loss was tested to be -29dB around the central frequency.

Upregulation of chemokine CXCL1/KC by leptospiral membrane lipoprotein preparation in renal tubule epithelial cells.

We have previously shown that leptospiral membrane lipoprotein preparation (LMLP) extracted from pathogenic Leptospira santarosai serovar Shermani stimulates the secretion of pro-inflammatory mediators in renal tubule epithelial cells, and implicated its role in the initiation of tubulointerstitial nephritis. Renal tubulointerstitial injury is characterized by inflammatory cell infiltrate; however, the stimuli for leukocyte recruitment are not fully understood. Initial studies by cytokine protein array analysis revealed significant upregulation of neutrophil-chemoattractant keratinocyte-derived chemokine (CXCL1/KC) at nanogram range of LMLP stimulation in cultured murine proximal tubule cells (PTCs). As PTCs express Toll-like receptors (TLRs), this study investigated the roles of TLR signaling pathways in PTCs stimulated by LMLP and its relation to CXCL1/KC secretion. The LMLP stimulated the early secretion of CXCL1/KC and enhanced the level of TLR2 mRNA expression in PTCs through time- and dose-dependent effect. The LMLP-stimulated secretion of human growth-related oncogene alpha, a functional homolog to murine KC, in TLR-defective human embryonic kidney 293 cells transiently transfected with TLR2-expressing plasmids and the response was augmented by coexpression of TLR1 and TLR2. Moreover, silencing of TLR2, myeloid differentiation factor 88, and TNF receptor-associated factor 6 with specific small interfering RNA significantly reduces the response caused by LMLP in PTCs. The LMLP-stimulated CXCL1/KC secretion was also significantly reduced by pre-incubating PTCs with a specific p38 inhibitor. These results indicate that LMLP stimulates the production of CXCL1/KC to recruit polymorphonuclear neutrophils at the site of inflammation through a TLR2-mediated pathway in renal tubule cells.

Toll-like receptor 2 mediates early inflammation by leptospiral outer membrane proteins in proximal tubule cells.

Tubulointerstitial nephritis is a cardinal renal manifestation in leptospirosis and LipL32, the major lipoprotein component of leptospiral outer membrane proteins (OMPs), induces a robust inflammatory response in cultured renal proximal tubule cells through a nuclear factor-kappaB-related pathway. Here, we investigated whether Toll-like receptor (TLR), known to play a pivotal role in innate immunity, could mediate the inflammatory response induced by leptospiral OMPs in renal proximal tubule cells. TLR expression was analyzed by flow cytometry and indirect immunofluorescence in cultured mouse proximal tubule (pyruvate kinase simian virus 40-proximal straight (PKSV-PR)) cells. Reverse transcription-competitive polymerase chain reaction and enzyme-linked immunosorbent assay were undertaken to analyze the inducible effects of inducible nitric oxide synthase (iNOS) and monocyte chemoattractant protein-1 (MCP-1 also termed CCL2) by pathogenic and non-pathogenic leptospiral OMPs and recombinant lipoproteins in either PKSV-PR cells or TLR-transfected human embryonic kidney (HEK) 293 cells. Anti-TLR antibodies were used for blocking experiments. Leptospira santarosai serovar Shermani OMPs and LipL32 induced a significant increase in TLR2 but not TLR4 expression in PKSV-PR cells. The increase in iNOS and CCL2/MCP-1 mRNA expressions could be prevented by an anti-TLR2 antibody, but not by an anti-TLR4 antibody. Furthermore, leptospiral OMPs stimulated both CCL2/MCP-1 mRNA and secreted protein in transfected HEK 293 cells with a TLR2-expressing plasmid, but had no effect in cells with a TLR4-expressing plasmid. In conclusion, these findings indicate that the stimulation of iNOS and CCL2/MCP-1 caused by pathogenic leptospiral OMPs, in particular LipL32, in proximal tubule cells requires TLR2 for the early inflammatory response.

Evaluation of lig-based conventional and real time PCR for the detection of pathogenic leptospires.

Leptospirosis is globally important infectious disease affecting almost all mammals. Pathogenic Leptospira encodes immunoglobulin-like protein (Lig) that is found to express only during infection. We report the development of conventional and real time PCR assays targeting lig genes of leptospires for the early diagnosis of leptospirosis. Sensitivity of the newly designed Lig1/Lig2 primers for conventional PCR was compared with previously published primers LP1/LP2 and G1/G2. G1/G2 primers amplified the target DNA from all the serovars including non-pathogenic Leptospira biflexa whereas LP1/LP2 and Lig1/Lig2 primers amplified only pathogenic leptospires. Diagnostic PCR assay was also developed for the detection of pathogenic Leptospira interrogans in urine samples. We obtained the highest sensitivity in PCR using our Lig1/Lig2 primers with a detection of 6 leptospires. A rapid and sensitive lig-based real time PCR assay was also developed with a detection range of 10-10(7) gene copies. To evaluate the early diagnosis for leptospirosis, we compared the culture with conventional and real time PCR for the detection of spirochetes in experimentally infected hamsters during a time-course study. Culture of infected hamster tissues detected the presence of leptospires from Day 2 of infection but not on the day of infection or Day 1, whereas conventional PCR and real time PCR detected the leptospires from the day of infection. Hence, conventional and real time PCR with lig primers would be a sensitive and rapid tool for early diagnosis of leptospirosis.

A seroepidemiology study of varicella among children aged 0-12 years in Taiwan.

The epidemiology pattern of varicella appears to vary among regions with different climates, population densities, and degrees of development. This study investigated the age-specific varicella zoster virus (VZV) seroprevalence in children aged 0 to 12 years in Taiwan and compared these seroprevalences between free and private vaccination areas. Residual sera were collected from 13 hospitals with 1,401 valid samples. Immunoglobulin G antibodies to VZV were measured by enzyme-linked immunosorbent assay. Parents of 656 children answered questions about the varicella incidence and varicella vaccination history of their children. In the 8-12 year-olds, the seroprevance ranged between 88.0-93.8% in northern, central, and eastern, while it was only 76.1% in southern Taiwan. The seroprevalence of children 0-5 years old were significantly different between free and private vaccination areas. Seropositive children who reported no history of varicella or receiving varicella vaccine accounted for 26.1-59.3% of the total positive cases. Our findings suggest the possible effects of climate, geographical conditions, and lifestyle on the seroepidemiology of VZV in Taiwan. The efforts of implementing a varicella vaccination program in Taiwan should focus on reaching high levels of coverage.

Protective effects of oral microencapsulated Mycoplasma hyopneumoniae vaccine prepared by co-spray drying method.

The efficacy of Mycoplasma hyopneumoniae oral vaccine was investigated in microsphere dosage form. A co-spray drying process was used to apply an encapsulating material, Eudragit L30 D-55, to microspheres containing Mycoplasma hyopneumoniae antigens. The microspheres were generally effective (>93%) with protein release at pH 7.4, but almost none were released at pH 1.2, for 3 hr in an in vitro dissolution test. An SPF-swine model was used to evaluate the effectiveness of the microspheres as an oral vaccine, and the related immune responses. The serum's systemic IgG against M. hyopneumoniae was evoked by ELISA analysis, after a 2nd immunization of all pigs. The vaccinated groups' mean lesion score was significantly lower after the Mycoplasma hyopneumoniae challenge than that of the nonvaccinated/challenged groups (P<0.05). This study strongly suggests that the oral microspheres vaccine prepared by a co-spray drying method can provide effective protection against M. hyopneumoniae infection in pigs.

In vivo and in vitro comparisons of spray-drying and solvent- evaporation preparation of microencapsulated Mycoplasma hyopneumoniae for use as an orally administered vaccine for pigs.

OBJECTIVE: To evaluate the efficacy of an orally administered vaccine of Mycoplasma hyopneumoniae that was prepared by spray drying or solvent evaporation. ANIMALS: Thirty 6-week-old, crossbred, specific-pathogen-free (SPF) pigs. PROCEDURE: Pigs were randomly allocated into 5 groups and housed in an SPF facility. Pigs in 2 groups (groups AQ and CAP) were fed M hyopneumoniae enteric-coated vaccine on days 0, 10, and 20. A third group (group IM) received an IM injection of M hyopneumoniae vaccine with aluminium hydroxide as an adjuvant on days 0, 10, and 20. The last 2 groups (non-vaccinated-challenged [NV-C] and nonchallenged [NC]) were fed a sham treatment. All 24 pigs in groups AQ, CAFP IM, and NV-C were challenge exposed with 5 ml of a 10% pneumonic lung suspension administered on day 40 via intubation of the trachea. All pigs were slaughtered and the lungs removed and examined for lesions on day 68. RESULTS: In vitro studies indicated that these 2 microencapsulation techniques formed an effective shell and protected mycoplasmal antigen from gastric acid. Results of inoculation and challenge tests indicated that microencapsulated M hyopneumoniae were sufficiently potent to induce an immune response and provide good protection. CONCLUSIONS AND CLINICAL RELEVANCE: Orally administered microencapsulated M hyopneumoniae vaccines induced an immune response and reduced the severity of lung lesions in challenge-exposed pigs. Results suggest that this novel method can be applied to other antigens, because the spray-drying process yielded an orally administered M hyopneumoniae vaccine that induced a good immune response.

Leptospirosis renal disease.

Leptospirosis is a re-emerging infectious disease, affecting both animals and humans worldwide. Multiple organ involvement may be encountered in leptospirosis, and early renal involvement is very common, characterized by tubulo-interstitial nephritis and tubular dysfunction. All 12 patients diagnosed in Chang Gung Memorial Hospital (Taiwan) between 1997 and 1999 had acute renal failure, with five patients requiring dialysis. Leptospira shermani is the main serovar encountered in Taiwan, and penicillin may dramatically rescue patients from multiple organ failure provided it is given early. To understand the mechanism behind tubular injuries by leptospira infection, outer membrane proteins (OMPs) extracted from pathogenic leptospira were given to tubular cells in culture. Our in vitro experiment showed that OMPs of pathogenic leptospira activate nuclear NFkappaB binding and stimulate downstream inducible nitric oxide (iNOS), monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-alpha) expression. These results indicate that leptospiral infection may induce tubulo-interstitial nephritis through a toxic component in the outer membrane followed by expression of inflammatory genes.

Negative incidence of Lyme disease-related Borrelia spp. in Alishan, Taiwan.

To investigate the prevalence of Lyme disease-related Borrelia species, wild rodents were captured around Yushan National Park and Alishan Forest Recreation Area Park in Taiwan 2,000 to 3,000 meters above sea level. Borrelia was not isolated from 67 small mammals of 7 species. Sera from rodents showed no positive reactivity against whole cell antigens of B. garinii, B. afzelii or B. valaisiana by ELISA. These results suggested that Lyme disease is not endemic to the Alishan area.

Genetic variation of the nucleocapsid genes of waterfowl parvovirus.

Duck parvovirus (DPV) and Goose parvovirus (GPV) isolated from infected waterfowls with Derzsy's disease in the year 1999 were identified by polymerase chain reaction and sequencing. The nucleotide sequences of their viral capsid proteins (VPs) show that they share 77% similarity at the DNA, and 84.6% at the protein level. The most variable region between DPV and GPV resides in the N-terminal of VP2 before the initiation codon of VP3 with 35% (19/54) amino acids divergence. Viral capsid protein sequences diverge 4.1 to 4.4% among 1990-99 isolated strains. Variant amino acids cluster in the common regions of VP3 at residues 203-266 and 482-534 which overlaps with the regions proposed to expose on the outer surfaces of parvoviral particles, implying that selective pressure from host immune system might play a part. These data provide useful information for antigenic epitope prediction. This study also reveal the presence of conserved strain-specific residues in VPs and these residues seldom vary among different viral isolates, suggesting that they might be functionally important and worth further investigation.

Characterization and identification of Borrelia isolates as Borrelia valaisiana in Taiwan and Kinmen Islands.

Eleven pure cultures of Borrelia from 3 species of wild rodents (Apodemus agrarius, Mus formosanus, Rattus losea) captured in Taichung, located in the center of Taiwan island, and on Kinmen Island were characterized. Five isolates showed restriction fragment length polymorphism (RFLP) patterns of 5S-23S rRNA gene intergenic spacer sequences identical to those of strains 5MT and 10MT, identified as Borrelia valaisiana, which were isolated in the southern tip of South Korea. Although the remaining six isolates showed novel RFLP patterns, these isolates showed more similarity to members of B. valaisiana from Korea, Japan and Europe based on 16S rRNA gene and flagellin gene sequences. This led us to speculate that transmission and proliferation of this type of borrelia occurred between Taiwan and the southern part of South Korea.

Leptospirosis: an ignored cause of acute renal failure in Taiwan.

Leptospirosis, caused by a spirochete, is the most common zoonosis in domestic or wild animals. Animals excrete infected urine in soil or water and may cause human infections through abrased wound, mucosa, conjunctiva, or by swallowing contaminated water. Clinical presentations of leptospirosis are mostly subclinical. Five to ten percent of leptospirosis are fatal, causing fever, hemorrhage, jaundice, and acute renal failure (Weil's syndrome). Leptospirosis has been ignored as a cause of acute renal failure in Taiwan. We report two patients with leptospirosis who presented with high fever, abdominal pain, jaundice, and acute renal failure. Patient 1 died on day 12 of admission of multiple organ failure associated with pancytopenia, hypogammaglobulinemia, and reactive hemophagocytosis. Leptospirosis was recognized after death. Patient 2 was admitted with similar presentations 2 weeks later. Penicillin and doxycycline were given early in the course, and azotemia, jaundice, respiratory failure, and aseptic meningitis gradually improved. Renal biopsy showed interstitial nephritis. Several tubular clearance tests showed proximal tubular defect with severe bicarbonate wasting (FeHCO3- 20.9%) and incomplete type II renal tubular acidosis without affecting the distal nephron. After 80 days of treatment, this patient was discharged with recovery of conscious level and renal function. This is the first leptospirosis patient with detailed tubular functional and morphological studies of the kidney. Diagnosis of leptospirosis was made by microscopic agglutination test (MAT) for antibody to leptospira and by polymerase chain reaction (PCR) for leptospira DNA in blood and urine (interrogans serogroup australis in case 1 and Leptospira borgpetersenii serogroup ballum in case 2). Because active surveillance has resulted in 13 cases diagnosed as leptospirosis islandwide thereafter, underestimation and ignorance of leptospirosis as a cause of acute renal failure may occur in Taiwan. Therefore, an area with a low leptospirosis incidence may actually have a very high incidence. Leptospirosis should be suspected in febrile patients with jaundice and renal failure when pathogens cannot be identified by traditional culture for microorganisms.

Two-step PCR in the evaluation of antibiotic treatment for Ehrlichia platys infection.

We evaluated the feasibility of using the two-step polymerase chain reaction (PCR) in determining the withdrawal time of antibiotic treatment for Ehrlichia platys infection. We also present experimental evidence of a dog remaining a carrier after treatment with tetracycline. Canine infectious cyclic thrombocytopenia (CICT) was induced in 3 dogs by intravenous inoculation of blood infected with E. platys. Tetracycline was administered to one of the dogs for 2 weeks when parasitemia appeared. Although the hematologic abnormality of cyclic thrombocytopenia soon disappeared, a few parasitized platelets reappeared after the withdrawal of treatment, and the dog thus remained as a carrier. The other dogs were treated with doxycycline when parasitemic episodes first developed. The durations of antibiotic regimens were determined by the results of two-step PCR in which the 16S rDNA of E. platys was amplified from blood samples. Doxycycline was withdrawn after 8 days of treatment, and the follow-up monitoring continued for 3 weeks. The platelet counts of the 2 dogs remained within the normal range, and the etiologic agent of CICT was not found either by Giemsa staining or by the two-step PCR, indicating complete elimination of the agent.

Sequence diversity of a gene encoding a putative primary sigma factor among Borrelia burgdorferi sensu lato strains.

Twenty-six strains of Borrelia burgdorferi sensu lato were subjected to polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis for assessing the sequence divergence of rpoD gene encoding the primary sigma factor. Four and five RFLP patterns were observed from two fragments of rpoD gene. Sequence analysis of a subgenic fragment covering region 1 through 4 from 13 strains of Borrelia burgdorferi s. 1. revealed that 21 of 450 deduced amino acid residues were diverged. These results indicate that the sequence heterogeneity of rpoD is present in different strains of Borrelia burgdorferi s. 1., and agreed well with the current classification of genospecies.

Specific amplification of Ehrlichia platys DNA from blood specimens by two-step PCR.

A two-step PCR method for diagnosis of canine infectious cyclic thrombocytopenia was established. Three primers derived from the 16S and rRNA gene sequence were used to amplify genomic DNA specifically from Ehrlichia platys. Two-round amplification with DNA templates prepared from E. platys-infected blood specimens produced 742- and 385-bp fragments, but these products were not found when an Ehrlichia canis-infected blood sample and Escherichia coli were used. This method, for which the minimum detectable copy number in the blood specimen was estimated to be five ehrlichia inclusion within platelets, is more sensitive than single PCR amplification. These results demonstrate that this two-step PCR is highly sensitive and efficient for detecting the etiologic agent of canine infectious cyclic thrombocytopenia in blood. The same technique was applied to blood specimens collected from a dog inoculated with E. platys. Amplification of the target DNA fragments was observed with blood collected on the fifth day after inoculation, which indicates that this method is also feasible for early diagnosis of E. platys infection.

Isolation of gE gene deleted pseudorabies virus by using a gE specific monoclonal antibody.

A simple, convenient method employing a gE-specific neutralizing monoclonal antibody (mAb; FTpn3) for isolation of the gE gene-deleted recombinant pseudorabies virus (PRV) is described. FTpn3 secreting hybridoma was obtained by fusing PRV-immunized BALB/c splenocytes with myeloma cells. To construct gE gene deleted PRV, a 5.7 kbp DNA fragment with deletion of the gE gene was engineered and cloned. The plasmid was then used for cotransfecting Vero cells with wild-type PRV genome. The resulting viruses were subjected to FTpn3 neutralization. The FTpn3 resistant virus was isolated and plaque purified further. By DNA fingerprinting and Western blotting analysis, the virus resistant to FTpn3 neutralization was proved to be the gE-deleted recombinant virus.

Canine infectious cyclic thrombocytopenia found in Taiwan.

Here were report the first canine infectious cyclic thrombocytopenia (CICT) found in Taiwan. Platelet-specific inclusions were detected in the blood smear of a military working dog. To identify the etiologic agent, the patient's blood was transmitted to three six-month-old German Shepherd dogs. The Ehrlichia platys-like inclusions were observed six to eight days after inoculation. Indirect fluorescent antibody test showed that the serum from the patient reacted specifically with the microorganisms within the platelets. Typical hematologic manifestations of E. platys infection, cyclic parasitemia and concomitant thrombocytopenia, were observed in these dogs. The prevalence of CICT in north Taiwan was also studied, and the incidence was 8.9% (4 out of 45) in civilian dogs and 97.1% (34 out of 35) in dogs from a heavily tick infested kennel.