Ground-penetrating radar (GPR) responses for sub-surface salt contamination and solid waste: modeling and controlled lysimeter studies.

The assessment of polluted areas and municipal solid waste (MSW) sites using non-destructive geophysical methods is timely and much needed in the field of environmental monitoring and management. The objectives of this study are (i) to evaluate the ground-penetrating radar (GPR) wave responses as a result of different electrical conductivity (EC) in groundwater and (ii) to conduct MSW stratification using a controlled lysimeter and modeling approach. A GPR wave simulation was carried out using GprMax2D software, and the field test was done on two lysimeters that were filled with sand (Lysimeter-1) and MSW (Lysimeter-2). A Pulse EKKO-Pro GPR system with 200- and 500-MHz center frequency antennae was used to collect GPR field data. Amplitudes of GPR-reflected waves (sub-surface reflectors and water table) were studied under different EC levels injected to the water table. Modeling results revealed that the signal strength of the reflected wave decreases with increasing EC levels and the disappearance of the subsurface reflection and wave amplitude reaching zero at higher EC levels (when EC >0.28 S/m). Further, when the EC level was high, the plume thickness did not have a significant effect on the amplitude of the reflected wave. However, it was also found that reflected signal strength decreases with increasing plume thickness at a given EC level. 2D GPR profile images under wet conditions showed stratification of the waste layers and relative thickness, but it was difficult to resolve the waste layers under dry conditions. These results show that the GPR as a non-destructive method with a relatively larger sample volume can be used to identify highly polluted areas with inorganic contaminants in groundwater and waste stratification. The current methods of MSW dumpsite investigation are tedious, destructive, time consuming, costly, and provide only point-scale measurements. However, further research is needed to verify the results under heterogeneous aquifer conditions and complex dumpsite conditions.

Tumour-suppressive function of SIRT4 in human colorectal cancer.

BACKGROUND: SIRT4, which is localised in the mitochondria, is one of the least characterised members of the sirtuin family of nicotinamide adenine dinucleotide-dependent enzymes that play key roles in multiple cellular processes such as metabolism, stress response and longevity. There are only a few studies that have characterised its function and assessed its clinical significance in human cancers. METHODS: We established colorectal cancer cell lines (SW480, HCT116, and HT29) overexpressing SIRT4 and investigated their effects on proliferation, migration and invasion, as well as E-cadherin expression, that negatively regulates tumour invasion and metastases. The associations between SIRT4 expression in colorectal cancer specimens and clinicopathological features including prognosis were assessed by immunohistochemistry. RESULTS: SIRT4 upregulated E-cadherin expression and suppressed proliferation, migration and invasion through inhibition of glutamine metabolism in colorectal cancer cells. Moreover, SIRT4 expression in colorectal cancer decreased with the progression of invasion and metastasis, and a low expression level of SIRT4 was correlated with a worse prognosis. CONCLUSIONS: SIRT4 has a tumour-suppressive function and may serve as a novel therapeutic target in colorectal cancer.

Fatal tracheal aspergillosis during rituximab combined chemotherapy for diffuse large B-cell lymphoma that developed after lung transplantation.

Invasive tracheal aspergillosis (ITA) is an infection that is unique to patients who have undergone lung transplantation (LT). Although the activity of this disease often appears on imaging, we encountered a case of ITA that became exacerbated, despite few computed tomography (CT) findings, during rituximab combined chemotherapy for diffuse large B-cell lymphoma. ITA developed during immunosuppressive therapy after LT. Because CT findings may show false-negative results, bronchoscopy is recommended for such cases.

MicroRNA-1246 expression associated with CCNG2-mediated chemoresistance and stemness in pancreatic cancer.

BACKGROUND: Pancreatic cancer has a poor prognosis because of its high refractoriness to chemotherapy and tumour recurrence, and these properties have been attributed to cancer stem cells (CSCs). MicroRNA (miRNA) regulates various molecular mechanisms of cancer progression associated with CSCs. This study aimed to identify the candidate miRNA and to characterise the clinical significance. METHODS: We established gemcitabine-resistant Panc1 cells, and induced CSC-like properties through sphere formation. Candidate miRNAs were selected through microarray analysis. The overexpression and knockdown experiments were performed by evaluating the in vitro cell growth and in vivo tumourigenicity. The expression was studied in 24 pancreatic cancer samples after laser captured microdissection and by immunohistochemical staining. RESULTS: The in vitro drug sensitivity of pancreatic cancer cells was altered according to the miR-1246 expression via CCNG2. In vivo, we found that miR-1246 could increase tumour-initiating potential and induced drug resistance. A high expression level of miR-1246 was correlated with a worse prognosis and CCNG2 expression was significantly lower in those patients. CONCLUSIONS: miR-1246 expression was associated with chemoresistance and CSC-like properties via CCNG2, and could predict worse prognosis in pancreatic cancer patients.

Amorfrutin B is an efficient natural peroxisome proliferator-activated receptor gamma (PPARγ) agonist with potent glucose-lowering properties.

AIMS/HYPOTHESIS: The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) is an important gene regulator in glucose and lipid metabolism. Unfortunately, PPARγ-activating drugs of the thiazolidinedione class provoke adverse side effects. As recently shown, amorfrutin A1 is a natural glucose-lowering compound that selectively modulates PPARγ. In this study we aimed to characterise, in vitro, a large spectrum of the amorfrutins and similar molecules, which we isolated from various plants. We further studied in vivo the glucose-lowering effects of the so far undescribed amorfrutin B, which featured the most striking PPARγ-binding and pharmacological properties of this family of plant metabolites. METHODS: Amorfrutins were investigated in vitro by binding and cofactor recruitment assays and by transcriptional activation assays in primary human adipocytes and murine preosteoblasts, as well as in vivo using insulin-resistant high-fat-diet-fed C57BL/6 mice treated for 27 days with 100 mg kg(-1) day(-1) amorfrutin B. RESULTS: Amorfrutin B showed low nanomolar binding affinity to PPARγ, and micromolar binding to the isotypes PPARα and PPARß/δ. Amorfrutin B selectively modulated PPARγ activity at low nanomolar concentrations. In insulin-resistant mice, amorfrutin B considerably improved insulin sensitivity, glucose tolerance and blood lipid variables after several days of treatment. Amorfrutin B treatment did not induce weight gain and furthermore showed liver-protecting properties. Additionally, amorfrutins had no adverse effects on osteoblastogenesis and fluid retention. CONCLUSIONS/INTERPRETATION: The application of plant-derived amorfrutins or synthetic analogues thereof constitutes a promising approach to prevent or treat complex metabolic diseases such as insulin resistance or type 2 diabetes.

miR-320c regulates gemcitabine-resistance in pancreatic cancer via SMARCC1.

BACKGROUND: Gemcitabine-based chemotherapy is the standard treatment for pancreatic cancer. However, the issue of resistance remains unresolved. The aim of this study was to identify microRNAs (miRNAs) that govern the resistance to gemcitabine in pancreatic cancer. METHODS: miRNA microarray analysis using gemcitabine-resistant clones of MiaPaCa2 (MiaPaCa2-RGs), PSN1 (PSN1-RGs), and their parental cells (MiaPaCa2-P, PSN1-P) was conducted. Changes in the anti-cancer effects of gemcitabine were studied after gain/loss-of-function analysis of the candidate miRNA. Further assessment of the putative target gene was performed in vitro and in 66 pancreatic cancer clinical samples. RESULTS: miR-320c expression was significantly higher in MiaPaCa2-RGs and PSN1-RGs than in their parental cells. miR-320c induced resistance to gemcitabine in MiaPaCa2. Further experiments showed that miR-320c-related resistance to gemcitabine was mediated through SMARCC1, a core subunit of the switch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex. In addition, clinical examination revealed that only SMARCC1-positive patients benefited from gemcitabine therapy with regard to survival after recurrence (P=0.0463). CONCLUSION: The results indicate that miR-320c regulates the resistance of pancreatic cancer cells to gemcitabine through SMARCC1, suggesting that miR-320c/SMARCC1 could be suitable for prediction of the clinical response and potential therapeutic target in pancreatic cancer patients on gemcitabine-based therapy.

Effect of highly lipolyzed goat cheese on HL-60 human leukemia cells: antiproliferative activity and induction of apoptotic DNA damage.

To establish cheese as a dairy product with health benefits, we embarked on examining the multifunctional role of cheeses, especially in the field of cancer prevention. The current study was designed to investigate whether different types of commercial goat cheeses may possess antiproliferative activity, using an HL-60 human promyelocytic leukemia cell line as a cancer cell model. Among 11 cheese extracts tested at 500µg/mL, 6 (Crottin de Chavignol, Pouligny Saint-Pierre, Chabichou du Poitou, Valencay, Kavli, and Sainte-Maure de Touraine) resulted in a significant decrease of cell viability, which is consistent with a decrease in viable cell number. Compared with the half-maximal inhibitory concentration (IC(50)) value of individual cheeses in cellular proliferation assays, the Pouligny Saint-Pierre extract showed strong inhibition. Incubation of cells in the presence of Pouligny Saint-Pierre extract resulted in induction of cellular morphological changes and apoptotic DNA fragmentation as well as expression of the active form of caspase-3 protein. Based on the quantification of the ratio of free fatty acids to triglycerides in different cheese samples, a significant correlation was detected between lipolytic ripeness and IC(50) values for antiproliferative capacity tested in HL-60 cells. Collectively, these results support a potential role of highly lipolyzed goat cheeses in the prevention of leukemic cell proliferation.

Computerised 3-D anatomical modelling using plastinates: an example utilising the human heart.

Computerised modelling methods have become highly useful for generating electronic representations of anatomical structures. These methods rely on crosssectional tissue slices in databases such as the Visible Human Male and Female, the Visible Korean Human, and the Visible Chinese Human. However, these databases are time consuming to generate and require labour-intensive manual digitisation while the number of specimens is very limited. Plastinated anatomical material could provide a possible alternative to data collection, requiring less time to prepare and enabling the use of virtually any anatomical or pathological structure routinely obtained in a gross anatomy laboratory. The purpose of this study was to establish an approach utilising plastinated anatomical material, specifically human hearts, for the purpose computerised 3-D modelling. Human hearts were collected following gross anatomical dissection and subjected to routine plastination procedures including dehydration (-25(o)C), defatting, forced impregnation, and curing at room temperature. A graphics pipeline was established comprising data collection with a hand-held scanner, 3-D modelling, model polishing, file conversion, and final rendering. Representative models were viewed and qualitatively assessed for accuracy and detail. The results showed that the heart model provided detailed surface information necessary for gross anatomical instructional purposes. Rendering tools facilitated optional model manipulation for further structural clarification if selected by the user. The use of plastinated material for generating 3-D computerised models has distinct advantages compared to cross-sectional tissue images.

Evaluation of pharyngeal swallowing pressure using high-resolution manometry during transoral surgery for oropharyngeal cancer.

BACKGROUND: Transoral robotic surgery is frequently described, driven by the desire to offer a less morbid alternative to chemoradiation. However, the objective evaluation of post-operative function has rarely been reported. Therefore, high-resolution manometry was used in this study to evaluate the impact of changes in peri-operative swallowing function on pharyngeal pressure events. METHODS: Ten patients with various stages of oropharyngeal cancer underwent transoral surgery. High-resolution manometry and videofluoroscopic swallow studies were performed before surgery and two months afterwards. The following parameters were obtained: velopharyngeal and mesopharyngeal post-deglutitive upper oesophageal sphincter pressures, velo-meso-hypopharyngeal contractile integral, upper oesophageal sphincter relaxation pressure, and pharyngeal velocity. RESULTS: There was no significant difference in pharyngeal pressure or contractile integral pre- versus post-operatively. However, pharyngeal velocity was significantly higher post-operatively than pre-operatively. CONCLUSION: High-resolution manometry showed that transoral surgery in patients without pre-operative dysphagia preserved pharyngeal constriction. However, transoral surgery might produce scar formation in the pharynx, which could lead to narrowing of the pharynx.

Required early complement activation in contact sensitivity with generation of local C5-dependent chemotactic activity, and late T cell interferon gamma: a possible initiating role of B cells.

Complement (C) is an important component of innate immunity, and was also shown recently to participate in induction of acquired B cell humoral immunity. In this study, we present evidence that C also participates in acquired T cell immunity. We found that C was involved in early events of the efferent elicitation phase of contact sensitivity (CS), and delayed-type hypersensitivity (DTH). Thus, CS and DTH were inhibited by administration of a C-blocker, soluble recombinant C receptor-1 (sCR1), when given 30 min before, but not 3 h after local antigen challenge. Among C components, local C5 were thought crucial to elicitation of CS, since local administration of anti-C5 monoclonal antibodies or locally injected C-depleting cobra venom factor also inhibited CS and DTH. These findings were consistent with our previous finding of the importance of C5 for CS elicitation, using congenitally C5-deficient mice. To dissect the mechanism of C dependence in CS, we demonstrated that locally increased early macrophage chemotactic activity (probably C5a) in evolving CS skin extracts, as well as late elaboration of IFN-gamma, were both inhibited by anti-C treatment. In addition, histological analysis showed that leukocyte recruitment into CS ear sites was similarly C-dependent. Furthermore, an initiating role of B cell-derived C-fixing immunoglobulin was suggested by demonstration of impaired CS responses in B cell-deficient mice. In summary, these results suggest that C was activated locally, perhaps via a B cell product, in an important early component of the stepwise events necessary to elicit CS, leading to local production of C5-dependent macrophage chemotactic activity and later IFN-gamma, and subsequently leading to cell infiltration, for development of T cell-dependent CS.

A simple and sensitive method for detection of Bacillus anthracis by loop-mediated isothermal amplification.

AIMS: To develop a rapid and simple system for detection of Bacillus anthracis using a loop-mediated isothermal amplification (LAMP) method and determine the suitability of LAMP for rapid identification of B. anthracis infection. METHODS AND RESULTS: A specific LAMP assay targeting unique gene sequences in the bacterial chromosome and two virulence plasmids, pXO1 and pXO2, was designed. With this assay, it was possible to detect more than 10 fg of bacterial DNA per reaction and obtain results within 30-40 min under isothermal conditions at 63 degrees C. No cross-reactivity was observed among Bacillus cereus group and other Bacillus species. Furthermore, in tests using blood specimens from mice inoculated intranasally with B. anthracis spores, the sensitivity of the LAMP assay following DNA extraction methods using a Qiagen DNeasy kit or boiling protocol was examined. Samples prepared by both methods showed almost equivalent sensitivities in LAMP assay. The detection limit was 3.6 CFU per test. CONCLUSIONS: The LAMP assay is a simple, rapid and sensitive method for detecting B. anthracis. SIGNIFICANCE AND IMPACT OF THE STUDY: The LAMP assay combined with boiling extraction could be used as a simple diagnostic method for identification of B. anthracis infection.

Adenovirus-mediated gene expression of the human c-FLIP(L) gene protects pig islets against human CD8(+) cytotoxic T lymphocyte-mediated cytotoxicity.

Cell-mediated immunity, especially of human CD8+ cytotoxic T lymphocytes (CTLs) is believed to have an important role in the long-term survival of pig islet xenografts. Protection against human CD8+ CTL cytotoxicity may reduce the direct damage to pig islets and enable long-term xenograft survival in pig-to-human islet xenotransplantation. We have previously reported that c-FLIP(S/L) genes, which are potent inhibitors of death receptor-mediated proapoptotic signals through binding competition with caspase-8 for recruitment to the Fas-associated via death domain (FADD), markedly suppress human CD8+ CTL-mediated xenocytotoxicity. In addition, the cytoprotective effects of c-FLIP(L) seem to be significantly stronger than those of c-FLIP(S). Accordingly, in the present study, expression of c-FLIP(L) was induced in intact pig islets by adenoviral transduction. Consequently, the cytoprotective capacity of the transgene in pig islets was examined in in vitro and in vivo exposure to human CD8+ CTLs. Cells from untransduced islets or mock islets were sensitive to CD8+ CTL-mediated lysis (59.3% +/- 15.9% and 64.0% +/- 8.9% cytotoxicity, respectively). In contrast, cells from pig islets transduced with the c-FLIP(L) gene were markedly protected from lysis (30.5% +/- 3.5%). Furthermore, prolonged xenograft survival was elicited from pig islets transduced with this molecule as assessed using an islet transplant model using the rat kidney capsule. Thus, these data indicate that intact pig islets can be transduced to express c-FLIP(L) with adenovirus. Pig islets expressing c-FLIP(L) are significantly resistant to human CTL killing and further exhibit beneficial effects to prolong xenograft survival.

In vivo controlling of cellular response to pig islet xenografts by adenovirus-mediated expression of either membrane-bound human FasL or human decoy Fas.

The critical problem with clinical islet transplantation for patients with type 1 diabetes is the severe shortage of human donors. Pig islet xenotransplantation has the potential to provide a virtually unlimited source of donor pancreata. However, our previous studies demonstrated that cell-mediated rejection, especially human CD8(+) cytotoxic T lymphocyte (CTL)-mediated cytotoxicity, remains a major obstacle for long-term islet xenograft survival. Moreover, we have demonstrated that the overexpression of either membrane-bound human FasL (mFasL) or human decoy Fas antigen (decoy Fas) in pig islets not only prevented CTL xenocytotoxicity in vitro, but also prolonged histological survival of pig islet xenografts in vivo. Therefore, the aim of the present study was to determine whether adenoviral transfer of these genes into pig islets ex vivo prior to transplantation had a beneficial effect on posttransplantation glycemic control of diabetic recipients. Isolated pig islets were transfected with adenovirus vector carrying complementary DNA (cDNA) of either mFasL or decoy Fas. The transfected islets were transplanted under the kidney capsule of diabetic recipient rats. Rats transplanted with either mFasL- or decoy Fas-transfected pig islet grafts showed significantly suppressed blood glucose levels from 12 hours to 18 hours posttransplantation compared with control groups transplanted with empty vector-transfected pig islets. Unfortunately, blood glucose levels of these groups were increased, with no significant difference observed at 24 hours posttransplantation. However, transgenic expression of these molecules with clinically tolerable amount of immunosuppressants may be more effective to achieve islet xenograft survival in the future.

Rapamycin induces autophagy in islets: relevance in islet transplantation.

Islet transplantation can provide insulin independence in patients with type 1 diabetes mellitus. However, islet allograft recipients exhibit a gradual decline in insulin independence, and only 10% do not require insulin at 5 years. This decline may reflect drug toxicity to islet beta cells. Rapamycin, a central immunosuppressant in islet transplantation, is a mammalian target of rampamycin inhibitor that induces autophagy. The relative contributions of autophagy in transplanted islets are poorly understood. Therefore, in the present study we sought to evaluate the effects of rapamycin on islet beta cells. Rapamycin treatment of islets resulted in accumulation of membrane-bound light chain 3 (LC3-II) protein, an early marker of autophagy. In addition, rapamycin treatment of isolated islets elicited not only reduction of viability but also downregulation of in vitro potency. To further examine the occurrence of autophagy in rapamycin-treated islets, we used GFP (green fluorescent protein)-LC3 transgenic mice that express a fluorescent autophagosome marker. The GFP-LC3 signals were markedly increased in rapamycin treated islets compared with control islets. In addition, to show improvement by blockade of autophagic signaling, islets were treated with rapamycin in the presence of 3-methyladenine, which inhibits autophagy. Thereafter, both islet viability and islet potency were dramatically improved. The number of GFP-LC3 dots clearly increased after 3-MA treatment. Thus, rapamycin treatment of islets induces autophagy in vitro. This phenomenon may contribute to the progressive graft dysfunction of transplanted islets. Therapeutically targeting this novel signaling may yield significant benefits for long-term islet survival.

Intracellular and extracellular remodeling effectively prevents human CD8(+)cytotoxic T lymphocyte-mediated xenocytotoxicity by coexpression of membrane-bound human FasL and pig c-FLIP(L) in pig endothelial cells.

Human CD8(+) cytotoxic T lymphocyte (CTL)-mediated cytotoxicity, which participates in xenograft rejection, is mediated mainly by the Fas/FasL apoptotic pathway. We previously developed methods to inhibit human CTL xenocytotoxicity by extracellular remodeling using overexpression of membrane-bound human FasL on pig xenograft cells, and by intracellular blockade of death receptor-mediated apoptotic signals, such as the Fas/FasL pathway using the pig c-FLIP(L) molecule. To investigate the cooperative effects of both membrane-bound FasL and pig c-FLIP(L), we cotransfected both genes into pig endothelial cells (PEC). The double remodeling with these molecules effectively prevented CD8(+) CTL killing. Although double transfectants and single high transfectants of either membrane-bound FasL or c-FLIP(L) gene displayed similar inhibition of CTL cytotoxicity, the expression levels of these 2 molecules in double transfectants were almost half the expression levels of single transfectants. Furthermore, to show in vivo prolongation of xenograft survival, we transplanted PEC transfectants under the rat kidney capsule. Prolonged survival was displayed by PEC double transfectant xenografts whereas those from either parental PEC or MOCK (vehicle control) were completely rejected by day 5 posttransplantation. These data suggested that intracellular and extracellular remodeling by coexpression of membrane-bound FasL and pig c-FLIP(L) in xenograft cells may prevent an innate cellular response to xenografts. The gene compatibility of these molecules to generate transgenic pigs may be sufficient to create a window of opportunity to facilitate long-term xenograft survival.

In vitro and in vivo prevention of human CD8+ CTL-mediated xenocytotoxicity by pig c-FLIP expression in porcine endothelial cells.

Overcoming cell-mediated immunity, especially of human CD8(+) CTLs, is important for the success of xenotransplantation. Our group has previously reported that the cytotoxicity of human CD8(+) CTLs against pig endothelial cells (PEC) is highly detrimental and mediated in major part by the Fas/FasL apoptotic pathway. Cellular FLICE inhibitory protein (c-FLIP) was originally identified as an inhibitor of death-receptor signaling through binding competition with caspase-8 for recruitment to Fas-associated via death domain (FADD). Two major c-FLIP variants result from alternative mRNA splicing: a short, 26-KDa protein (c-FLIP(S)) and a long, 55-KDa form (c-FLIP(L)). The cytoprotective effects of c-FLIP(S/L) in xenograft cells remain controversial. This study demonstrates that the overexpression of c-FLIP(S/L) genes markedly suppress human CD8(+) CTL-mediated xenocytotoxicity and, in addition, the cytoprotective effects of c-FLIP(L) appear to be significantly stronger than those of c-FLIP(S). Furthermore, to prove the prolonged effects of xenograft survival, PEC transfectants with c-FLIP(S/L) genes were transplanted under rat kidney capsules. Prolonged survival was elicited from FLIP(S/L) transfectants, whereas parental PEC was completely rejected through day 5, posttransplant. Thus, intracellular remodeling with the overexpression of c-FLIP(S/L) in xenograft cells may avoid innate cellular attacks against xenografts and facilitate long-term xenograft survival.

Transplantation of newborn thymus plus hematopoietic stem cells can rescue supralethally irradiated mice.

We attempted to rescue supralethally irradiated (SLI) mice by transplantation of hematopoietic stem cells (HSCs) plus thymus from variously aged donors (fetus, newborn and adult). Although the transplantations of these kinds of HSCs alone showed a very short survival, newborn liver cells (NLCs) (as the source of HSCs) plus newborn thymus (NT) transplantation markedly improved the survival rate. The transplantation attenuated severe damage in the small intestine, which is one of the major causes of death by SLI. In addition, the donor-derived CD4(+) T cells significantly increased with additional NT transplantation. The production of interleukin (IL)-7 and keratinocyte growth factor, which plays a crucial role in protection against radiation injury in the intestine, was the highest in NT. Finally, SLI mice that had received NLC plus IL-7(-/-) NT transplantation plus IL-7 injection showed improved survival, weight recovery and an elevated number of CD4(+) T cells compared with the mice that had received NLC plus IL-7(-/-) NT or plus IL-7 injection alone. These findings suggest that NLCs plus NT transplantation can rescue SLI mice most effectively, and that high production of IL-7 in NT plays a crucial role with induction of CD4(+) T cells.

Rapid detection of Brucella spp. by the loop-mediated isothermal amplification method.

AIMS: To develop a rapid and sensitive method for detecting Brucella spp. METHODS AND RESULTS: Two sets of six Brucella-specific primers for loop-mediated isothermal amplification (LAMP) were designed from the sequence of the Brucella abortus BCSP31 gene. The specificity and sensitivity were examined for six Brucella species (22 strains) and 18 non-Brucella species (28 strains). The LAMP assay was specific to Brucella spp. in 35 min at 63 degrees C and sensitive (detected 10 fg of genomic DNA). The assay was also applied for the detection of Brucella DNA in contaminated milk and infected mouse organs. CONCLUSIONS: We developed a sensitive and specific LAMP assay for Brucella spp., with the test appearing to be useful for the detection of the pathogen from clinical and food samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the development of LAMP for the detection of Brucella spp. As the LAMP assay can be performed at a constant temperature and its reactivity is directly observed with the naked eye without electrophoresis, our assay should be useful for the diagnosis of brucellosis as well as the detection of the bacteria in environmental or food samples.

Pig cellular FLICE-like inhibitory protein (c-FLIP) overexpression in pig xenograft cells induces resistance to human CD8+ cytotoxic T lymphocyte-mediated xenocytotoxicity.

Although the use of organs from alpha1,3-galactosyltransferase gene knockout pigs may prolong xenograft survival, resulting in overcoming antibody-mediated hyperacute rejection, pig xenografts will be destroyed directly by cell-mediated immunity, such as NK cells, macrophages, and CD8+ cytotoxic T lymphocytes (CTLs). Therefore, conquering cell-mediated immunity, especially of human CD8+ CTLs, is of particular importance to the success of long-term xenograft survival. We have previously reported that the cytotoxicity of human CD8+ CTLs is strong against pig endothelial cells (PEC) and mediated in major part by the Fas/FasL apoptotic pathway. Cellular FLICE inhibitory protein (c-FLIP) was originally identified as a potent inhibitor of death-receptor signaling through binding competition with caspase-8 for recruitment to Fas-associated via death domain (FADD). Two major c-FLIP variants result from alternative mRNA splicing: a short, 26-kDa protein (c-FLIP S) and a long, 55-kDa form (c-FLIP L). The present study demonstrated that overexpression of c-FLIP S/L genes in PEC markedly suppressed human CD8+ CTL-mediated xenocytotoxicity; moreover, the cytoprotective effects of c-FLIP L appeared to be significantly stronger than those of c-FLIP S. Furthermore, to prove the in vivo prolongation effects of xenograft survival, we transplanted PEC transfectants with c-FLIP(S/L) genes under the rat kidney capsule. Prolonged survival was displayed by xenografts of FLIP S/L PEC transfectants, whereas xenografts of parental PEC were completely rejected by day 5 posttransplantation. Thus, intracellular blocking of death receptor-mediated apoptotic signals by overexpression of c-FLIP S/L in xenograft cells may prevent innate cellular attacks against xenografts opening the window of opportunity for long-term xenograft survival.