Improved outcomes of islet autotransplant after total pancreatectomy by combined blockade of IL-1ß and TNFα.

The efficacy of islet transplant is compromised by a significant loss of islet mass posttransplant due to an innate inflammatory reaction. We report the use of a combination of etanercept and anakinra (ANA+ETA) to block inflammatory islet damage in 100 patients undergoing total pancreatectomy with islet autotransplant. The patients were divided into 3 groups: no treatment (control [CTL]), etanercept alone (ETA), or a combination of etanercept and anakinra (ANA+ETA). Peritransplant serum samples were analyzed for protein markers of islet damage and for inflammatory cytokines. Graft function was assessed by fasting blood glucose, basal C-peptide, secretory unit of islet transplant objects (SUITO) index, and hemoglobin A1c . Administration of both antiinflammatory drugs was well tolerated without any major adverse events. Reductions in interleukin-6, interleukin-8, and monocyte chemoattractant protein 1 were observed in patients receiving ANA+ETA compared with the CTL group, while also showing a modest improvement in islet function as assessed by basal C-peptide, glucose, hemoglobin A1c , and SUITO index but without differences in insulin dose. These results suggest that double cytokine blockade (ANA+ETA) reduces peritransplant islet damage due to nonspecific inflammation and may represent a promising strategy to improve islet engraftment, leading to better transplant outcomes.

Remnant living cells that escape cell loss in late-stage tumors exhibit cancer stem cell-like characteristics.

A balance between cell proliferation and cell loss is essential for tumor progression. Although up to 90% of cells are lost in late-stage carcinomas, the progression and characteristics of remnant living cells in tumor mass are unclear. Here we used molecular imaging to track the progression of living cells in a syngeneic tumor model, and ex vivo investigated the properties of this population at late-stage tumor. The piggyBac transposon system was used to stably introduce the dual reporter genes, including monomeric red fluorescent protein (mRFP) and herpes simplex virus type-1 thymidine kinase (HSV1-tk) genes for fluorescence-based and radionuclide-based imaging of tumor growth in small animals, respectively. Iodine-123-labeled 5-iodo-2'-fluoro-1-beta-D-arabinofuranosyluracil was used as a radiotracer for HSV1-tk gene expression in tumors. The fluorescence- and radionuclide-based imaging using the single-photon emission computed tomography/computed tomography revealed that the number of living cells reached the maximum at 1 week after implantation of 4T1 tumors, and gradually decreased and clustered near the side of the body until 4 weeks accompanied by enlargement of tumor mass. The remnant living cells at late-stage tumor were isolated and investigated ex vivo. The results showed that these living cells could form mammospheres and express cancer stem cell (CSC)-related biomarkers, including octamer-binding transcription factor 4, SRY (sex-determining region Y)-box 2, and CD133 genes compared with those cultured in vitro. Furthermore, this HSV1-tk-expressing CSC-like population was sensitive to ganciclovir applied for the suicide therapy. Taken together, the current data suggested that cells escaping from cell loss in late-stage tumors exhibit CSC-like characteristics, and HSV1-tk may be considered a theranostic agent for targeting this population in vivo.

First Report of Pepper mottle virus in Bell Pepper in Taiwan.

Bell pepper (Capsicum annuum L.) plants exhibiting systemic mild mosaic, vein yellowing, and leaf malformation were collected from Puli City in 2006. Double-antibody sandwich (DAS)-ELISA was used to test these samples for Chilli veinal mottle virus (ChiVMV) infection using polyclonal antibodies. In addition, Chenopodium quinoa, C. amaranticolor, and Nicotiana benthamiana plants were mechanically inoculated with sap extracted from collected samples. Ten days postinoculation, chlorotic local lesions were observed on inoculated leaves of C. quinoa and C. amaranticolor plants, whereas, systemic mosaic and foliar distortion symptoms were developed on upper leaves of N. benthamiana plants. The DAS-ELISA test showed that field-collected pepper samples and inoculated leaves of C. quinoa and C. amaranticolor were infected with ChiVMV, while N. benthamiana with mosaic symptoms did not react with ChiVMV antibodies. To confirm ChiVMV, field-collected samples as well as mechanically inoculated plants were tested by reverse transcription (RT)-PCR using the potyvirus degenerate primers Hrp5/Pot1 (2). Amplified RT-PCR products were cloned and sequenced. Sequence analysis of amplified fragments (1.4 kb) revealed that field-collected pepper samples were infected with ChiVMV and Pepper mottle virus (PepMoV). The DNA fragment amplified from C. quinoa and C. amaranticolor showed high (99.2%) sequence identities with the CP gene of ChiVMV (3) (GenBank Accession No. AM909717). However, amplicons obtained from N. benthamiana plants (GenBank Accession No. HQ329082) that showed mosaic symptoms showed 83.6% to 98.7% nucleotide identities with PepMoV (GenBank Accession Nos. AB126033, AF227728, AF440801, AF501591, EU586133, and M96425). Next, a pure isolate of PepMoV was established on N. benthamiana by mechanical inoculation of diluted plant sap obtained from a PepMoV-infected N. benthamiana plant. Bell pepper plants inoculated with the Taiwan isolate of PepMoV developed mosaic and leaf distortion symptoms. Antiserum against the PepMoV Taiwan isolate was subsequently prepared by immunizing rabbits with purified virus particles. Using the prepared antiserum and specific primers (1) to detect PepMoV, ChiVMV, and Pepper veinal mottle virus (PVMV), three viruses could be readily detected and differentiated from diseased bell peppers in the field. In a survey done in 2007, 18 of 33 pepper samples from southern Taiwan were found with mixed infections of PepMoV and ChiVMV, seven samples were infected with PepMoV and PVMV, five samples were infected with PVMV, and another three samples were infected with ChiVMV. To our knowledge, this is the first report of the occurrence of PepMoV in bell peppers in Taiwan. References: (1) Y. H. Cheng et al. Plant Dis. 93:107, 2009. (2) S. S. Pappu et al. Plant Dis. 82:1121, 1998. (3) W. S. Tsai et al. Plant Pathol. 58:408, 2008.

First Report of Pepper veinal mottle virus in Tomato and Pepper in Taiwan.

In May of 2006, samples from tomato plants (Solanum lycopersicum cv. Known-you 301) exhibiting necrotic symptoms on stems, petioles, and leaves were collected from Chiayi County, Taiwan. Double-antibody sandwich-ELISAs were performed using Cucumber mosaic virus, Tomato mosaic virus, Potato virus Y, Watermelon silver mottle virus, and Chilli veinal mottle virus (ChiVMV) polyclonal antibodies. Three of eight samples reacted with antibodies against ChiVMV but not with the others. Using the potyvirus degenerate primers (Hrp 5/Pot 1) (2), an expected 1.5-kb DNA fragment including the 3'-end of the NIb gene, the complete coat protein (CP) gene, and the 3'-nontranslatable region of the virus was amplified from total RNA isolated from these three samples by reverse transcription (RT)-PCR. A homology search in GenBank indicated that the new tomato-infecting virus in Taiwan belongs to Pepper veinal mottle virus (PVMV) since they shared >90% amino acid identity in the CP gene. A virus culture (Tom1) isolated from one of the diseased tomatoes was then established in Chenopodium quinoa and Nicotiana benthamiana and the CP gene was amplified and sequenced (GenBank Accession No. EU719647). Comparisons of the 807-nt CP gene with those of five PVMV isolates available in GenBank showed 81.5 to 93.1% nucleotide and 90.0 to 97.8% amino acid identity. Tom1 induced irregular necrotic lesions on stems, petioles, and leaves of tomato while inducing only mild mottle symptoms on pepper. Serological cross reaction between ChiVMV and PVMV has been observed previously (1,3) and also found in this study. To differentiate these two potyviruses by RT-PCR, primer pair CPVMVup/dw (5'-TATTC(T/C)TCAGTGTGG(A/T/C)T(T/C)CCACCAT and 5'-(T/C)C(A/T)C(A/T)(A/T/G)(A/T)AA(A/G)CCATAA(A/C)(A/C)ATA(A/G)T(T/C)T) was designed on the basis of the comparison of the CP gene and the 3'-nontranslatable region of the PVMV and ChiVMV. DNA fragments of 171 and 259 bp are expected to be amplified from ChiVMV and PVMV, respectively, by RT-PCR with primers CPVMVup/dw. In a field survey done in 2006, samples from diseased peppers (Capsicum annuum) that reacted with the polyclonal antibodies against ChiVMV were further identified by RT-PCR with primers CPVMVup/dw, indicating that both ChiVMV and PVMV infected pepper crops (Capsicum spp.) in Taiwan. A pepper isolate (Pep1) of PVMV was obtained from Nantou County through three times of single lesion passages on C. quinoa and then propagated on N. benthamiana. The CP gene of Pep1 was amplified and sequenced (GenBank Accession No. EU719646) and found to share 99.1% nucleotide and 100% amino acid identity with that of Tom1. Pep1 caused mild mottle symptoms on leaves of both tomato and pepper. To our knowledge, this is the first report of the presence of PVMV in Taiwan as well as in East Asia. References: (1) B. Moury et al. Phytopathology 95:227, 2005. (2) S. S. Pappu et al. Plant Dis. 82:1121, 1998. (3) W. S. Tsai et al. Plant Pathol. 58:408, 2008.

Identifying differentially expressed genes associated with metastasis of follicular thyroid cancer by cDNA expression array.

Patients with follicular thyroid carcinoma have a higher incidence of metastasis than papillary thyroid carcinoma when thyroid cancer is diagnosed. The cDNA expression array technology is utilized herein to profile differentially expressed genes from metastatic human follicular thyroid carcinoma and reveal new tumor markers as well as target genes for therapeutic intervention. Tissue samples were obtained during surgical resection of the thyroid follicular carcinoma and metastatic tissue in the brain of the same patient. Two identical Atlas human cDNA expression arrays were hybridized with 32P-labeled cDNA probes derived from RNA of either primary thyroid cancer or metastatic tissue. Parallel analysis of the hybridized signals allowed us to identify the alteration of gene expression in the metastasis process. Eighteen genes significantly overexpressed and 40 genes significantly underexpressed were identified in the metastatic thyroid cancer. Genes that displayed an altered expression were associated with the processes of cell cycle regulation, apoptosis, DNA damage response, angiogenesis, cell adhesion and mobility, invasion, and immune response. An expression profile of genes that are associated with metastasis process of follicular thyroid cancer was also discussed. Further investigation is required to understand the precise relationship between the altered expression of these genes and the metastasis process of follicular thyroid cancer.

Helicobacter pylori induces gene expression in human gastric cells identified by mRNA differential display.

Changes in gene expression patterns in gastric cells infected with Helicobacter pylori were characterized by means of mRNA differential display. Total RNA preparations were extracted from the H. pylori infected gastric cells and paired non-infected cells, and were probed with candidate clones identified after screening up to 6,000 mRNA species. Among them, four clones, 04G-1, 04G-2, 01G-1, and Cppa-2 show significant expression in the infected cells by Northern blot analysis, and they are 199 bp, 196 bp, 228 bp, and 276 bp in length, respectively. Database search revealed that nucleotide sequences of these clones share very low identity with any known sequence. These results indicate that H. pylori can significantly affect gene expression in gastric cells. Furthermore mRNA differential display can be used in pathogenesis studies to identify new genes in gastric cells in response to insults such as H. pylori.