Victor Fei Pang

Reduction of porcine reproductive and respiratory syndrome virus (PRRSV) infection in swine alveolar macrophages by porcine circovirus 2 (PCV2)-induced interferon-alpha.

Abstract Two common viral pathogens of swine, namely, porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV), were investigated in regard to their effects on monolayer cultures of swine alveolar macrophages (AMs). The purpose was to identify selected cellular changes and responses potentially associated with the clinical reactions of pigs infected with either or both of these viruses. Measurements included the (1) absolute and relative numbers of infected, viable, and apoptotic cells; (2) distribution of viral antigens; (3) levels of interferon-alpha (IFN-α) and tumor necrosis factor-alpha (TNF-α) produced and their association with the extent of virus-induced cytopathology. Four groups of AMs were studied, including mock-infected, PCV2 alone-infected (PCV2-A), PRRSV alone-infected (PRRSV-A), and PCV2 and PRRSV dually infected (PCV2/PRRSV) groups. The AMs of PCV2-A group had high antigen-containing rate without cell death. There was a marked increase in cell death and apoptosis in PRRSV-A group. However, a lower PRRSV-induced infectious rate, cell death, and apoptosis were seen in PCV2/PRRSV group. High levels of IFN-α production were detected in PCV2-infected groups, but not in mock-infected and PRRSV-A groups. The PRRSV-induced cytopathic effect (CPE) on MARC-145 cells or swine AMs was markedly reduced by pre-incubation of the cells with UV-treated or non-UV-treated supernatants of PCV2-infected AMs. In addition, the reduction in CPE was abolished when the supernatants of PCV2-infected AMs were pre-treated with a mouse anti-recombinant porcine IFN-α antibody. The results suggest that swine AMs were an important reservoir of PCV2; PCV2 infection reduced PRRSV infection and PRRSV-associated CPE in PCV2/PRRSV AMs; the reduction of PRRSV infection in AMs was mediated by IFN-α generated by PCV2 infection. The reduced PRRSV-associated CPE in AMs and increased pro-inflammatory cytokine production may lead to a more severe pneumonic lesion in those dually infected pigs.

Effects of porcine reproductive and respiratory syndrome virus (isolate tw91) on porcine alveolar macrophages in vitro.

To verify the role of porcine reproductive and respiratory syndrome virus (PRRSV) infection on pulmonary defense mechanisms, alterations in the viability, morphology, and various functions of porcine alveolar macrophages (AMs) were evaluated in vitro for 2-72 h after exposure to a Taiwan isolate, tw91, at a multiplicity of infection (MOI) of 0.1. A low but constant rate of infection, around 5%, was seen in AMs from the PRRSV-infected group throughout the study. When compared with a mock-infected group, AMs from the PRRSV-infected group had a significantly lower viability at 18-72 h post-infection (HPI) as determined by trypan blue dye exclusion. Also during this time period, the cells showed morphological changes, including rounding, bleb formation, and rupture. The phagocytic and microbicidal capacity of AMs against Candida albicans was significantly inhibited after 6 HPI. Although the total amount of superoxide anion (O2-) and hydrogen peroxide (H2O2) produced by the AMs was reduced after 18 and 12 HPI, respectively, the amount of production was enhanced in both reactive oxygen species on a per viable cell basis after 12 HPI. In contrast, the level of bioactive tumor necrosis factor alpha (TNF-alpha) secretion, either total or on a per viable cell basis, was markedly reduced soon after PRRSV infection, up to 36 HPI, followed by a rebound thereafter. Prostaglandin E2 (PGE2) production was enhanced, both in total and on a per viable cell basis, in the first 6 h of infection, especially at 2 HPI. However, it became lower than that of the control after 36 HPI. The results indicated that PRRSV infection could cause, directly and/or indirectly, not only death of AMs but also adverse alterations in their morphology and function, although some of the effects seemed to be reversible. Because AMs are crucial to the host against airborne pathogens, PRRSV infection may potentially predispose pigs to secondary pulmonary infections.

Diagnostic and therapeutic evaluation of 111In-vinorelbine-liposomes in a human colorectal carcinoma HT-29/luc-bearing animal model

Colorectal carcinoma is a highly prevalent and common cause of cancer in Taiwan. There is still no available cure for this malignant disease. To address this issue, we applied the multimodality of molecular imaging to explore the efficacy of diagnostic and therapeutic nanoradiopharmaceuticals in an animal model of human colorectal adenocarcinoma [colorectal cancer (CRC)] that stably expresses luciferase (luc) as a reporter. In this study, an in vivo therapeutic efficacy evaluation of dual-nanoliposome (100 nm in diameter) encaged vinorelbine (VNB) and (111)In-oxine on HT-29/luc mouse xenografts was carried out. HT-29/luc tumor cells were transplanted subcutaneously into male SCID mice. Multimodality of molecular imaging approaches including bioluminescence imaging (BLI), gamma scintigraphy, whole-body autoradiography (WBAR) and in vivo tumor growth tracing, histopathology and biochemistry/hematology analyses were applied on xenografted SCID mice to study the treatments with 6% polyethylene glycol (PEG) of (111)In-NanoX/VNB-liposomes. In vivo tumor growth tracing and BLI showed that tumor volume could be completely inhibited by the combination therapy with (111)In-VNB-liposomes and by chemotherapy with NanoX/VNB-liposomes (i.e., without Indium-111) (P<.01). The nuclear medicine images of gamma scintigraphy and WBAR also revealed the conspicuous inhibition of tumor growth by the combination therapy with (111)In-VNB-liposomes. Animal body weights, histopathology and biochemistry/hematology analyses were used to confirm the safety and feasibility of radiopharmaceuticals. A synergistic therapeutic effect on CRC xenografted SCID mice was proven by combining an Auger electron-emitting radioisotope (Indium-111) with an anticancer drug (VNB). This study further demonstrates the beneficial potential applications of multimodality molecular imaging as part of the diagnostic and therapeutic approaches available for the evaluation of new drugs and other strategic approaches to disease treatment.

Effects of aflatoxin and carotenoids on growth performance and immune response in mule ducklings.

The purpose of this study was to investigate if carotenoids could alleviate the adverse effects caused by aflatoxin with respect to growth performance and immune response. In two experiments, a total of 320 mule ducklings were assigned to 5 treatments, i.e. control, aflatoxin B(1) (AFB(1)) 200 ppb, AFB(1) +beta-carotene (BC) 200 ppm, AFB(1)+BC 400 ppm, and AFB(1)+astaxanthin (AS) 200 ppm. In experiment 1, the addition of beta-carotene or astaxanthin in the diet containing AFB(1) 200 ppb resulted in a significant decrease in average daily gain as compared with the control. AFB(1) 200 ppb alone and the addition of BC or AS on top of AFB(1) resulted in a significantly lower daily feed intake than for the control group. There were no significant differences in relative organ weights among treatment groups. Both treatments of BC 400 ppm and AS 200 ppm had significantly more macrophages harvested per duck than the control and AFB(1) 200 ppb treatments. However, there were no significant differences among treatments in percentages of phagocytotic macrophages and number of Candida albican phagocytized by phagocytotic macrophages. In experiment 2, blood biochemical parameters and antibody titers were evaluated. There were no significant differences among treatments in total bilirubin content and alkaline phosphatase activity in the serum or in antibody titers against fowl cholera. However, AFB(1) treatment had the highest activities of AST and ALT in the serum. The addition of BC 400 ppm on top of AFB(1) significantly reduced ALT activity as compared with the AFB(1) 200 ppb treatment. These results suggest that carotenoids could provide a slightly toxic alleviating effect on growth performance, enhance the chemotaxis ability of macrophages, and reduce ALT activity elevated by AFB(1).

Therapeutic Efficacy Evaluation of 111In-Labeled PEGylated Liposomal Vinorelbine in Murine Colon Carcinoma with Multimodalities of Molecular Imaging

In our previous studies using combined radioisotopes with chemotherapeutic liposomal drugs (i.e., 111In-labeled polyethylene glycol (PEG)ylated liposomal vinorelbine) we have reported possible therapeutic efficiency in tumor growth suppression. Nevertheless, the challenge remains as to whether this chemotherapy has a therapeutic effect as good as that of combination therapy. The goal of this study was to investigate the real therapeutic effectiveness of 6 mol% PEG 111In-vinorelbine liposomes via the elevation of the radiation dosage and reduction in the concentration of chemotherapeutic agents. Methods: Murine colon carcinoma cells transfected with dual-reporter genes (CT-26/tk-luc) were xenografted into BALB/c mice. The biodistribution was estimated to determine the drug profile and targeting efficiency of 111In-vinorelbine liposomes. Bioluminescence imaging and 18F-FDG small-animal PET were applied to monitor the therapeutic response after drug administration. The survival in vivo was estimated and linked with the toxicologic and histopathologic analyses to determine the preclinical safety and feasibility of the nanomedicine. Results: Effective long-term circulation of radioactivity in the plasma was achieved by 6 mol% PEG 111In-vinorelbine liposomes, and this dose showed significantly lower uptake in the reticuloendothelial system than that of 0.9 mol% PEG 111In-vinorelbine liposomes. Selective tumor uptake was represented by cumulative deposition, and the maximum accumulation was at 48 h after injection. The combination therapy exhibited an additive effect for tumor growth suppression as tracked by caliper measurement, bioluminescence imaging, and small-animal PET. Furthermore, an improved survival rate and reduced tissue toxicity were closely correlated with the toxicologic and histopathologic results. Conclusion: The results demonstrated that the use of 6 mol% PEG 111In-vinorelbine liposomes for passively targeted tumor therapy displayed an additive effect with combined therapy, not only by prolonging the circulation rate because of a reduction in the phagocytic effect of the reticuloendothelial system but also by enhancing tumor uptake. Thus, this preclinical study suggests that 6 mol% PEG 111In-vinorelbine liposomes have the potential to increase the therapeutic index and reduce the toxicity of the passively nanotargeted chemoradiotherapies.

Essential roles of caspases and their upstream regulators in rotenone-induced apoptosis

In the present study, we examined whether caspases and their upstream regulators are involved in rotenone-induced cytotoxicity. Rotenone significantly inhibited the proliferation of oral cancer cell lines in a dose-dependent manner compared to normal oral mucosal fibroblasts. Flow cytometric analysis of DNA content showed that rotenone treatment induced apoptosis following G2/M arrest. Western blotting showed activation of both the caspase-8 and caspase-9 pathways, which differed from previous studies conducted in other cell types. Furthermore, p53 protein and its downstream pro-apoptotic target, Bax, were induced in SAS cells after treatment with rotenone. Rotenone-induced apoptosis was inhibited by antioxidants (glutathione, N-acetylcysteine, and tiron). In conclusion, our results demonstrate significant involvement of caspases and their upstream regulators in rotenone-induced cytotoxicity.

Immunogenicity of recombinant GP5 protein of porcine reproductive and respiratory syndrome virus expressed in tobacco plant.

The aim of the study was to evaluate the immunogenicity of the ORF5-encoded major envelop glycoprotein 5 (GP5) of porcine reproductive and respiratory syndrome virus (PRRSV) expressed in tobacco plant as a potential pig oral vaccine in protection against PRRSV infection. Six-week-old PRRSV-free pigs were fed four times orally with 50g of chopped fresh GP5 transgenic tobacco leaves (GP5-T) (GP5 reaching 0.011% of total soluble protein) or wild-type tobacco leaves (W-T) each on days 0, 14, 28, and 42. Samples of serum, saliva, and peripheral blood mononuclear cells (PBMCs) were collected on days -1, 6, 13, 20, 27, 34, 41, and 48 after the initial oral vaccination. A similar vaccination-dependent gradual increase in the responses of serum and saliva anti-PRRSV total IgG and IgA, respectively, and in the levels of PRRSV-specific blastogenic response of PBMCs was seen in GP5-T-treated pigs; all statistically significant elevations occurred after the 2nd vaccination and were revealed after 20 days post-initial oral vaccination (DPIOV). Pigs fed on GP5-T also developed serum neutralizing antibodies to PRRSV at a titer of 1:4-1:8 after the 4th vaccination by 48 DPIOV. No detectable anti-PRRSV antibody responses and PRRSV-specific blastogenic response were seen in W-T-treated pigs. The present study has demonstrated that pigs fed on GP5-T could develop specific mucosal as well as systemic humoral and cellular immune responses against PRRSV. The results also support that transgenic plant as GP5-T can be an effective system for oral delivery of recombinant subunit vaccines in pigs.

Experimental T-2 toxicosis in swine: III. Morphologic changes following intravascular administration of T-2 toxin

The gross and microscopic changes in swine following a single intravascular (iv) dose of T-2 toxin are described and evaluated quantitatively. T-2 toxin, in 70% ethanol, was given iv at 0 (5 pigs), 0.6 (5 pigs), 1.2 (1 pig), 4.8 (5 pigs), or 5.4 (2 pigs) mg/kg to 40 to 60 kg female crossbred pigs. The 4.8 and 5.4 mg/kg group pigs died between 5 and 10.5 hr after treatment, while the 0, 0.6, and 1.2 mg/kg pigs were killed at 24, 24, and 12 hrs after treatment, respectively. Morphologic examination was performed at the gross and light microscopic levels. In addition, a quantitative evaluation of microscopic changes present in lymphoid tissues and intestinal tract was performed using a semiquantitative scoring system. Gross lesions in the T-2-treated pigs consisted of edema, congestion, and hemorrhage of the lymph nodes and pancreas; congestion and hemorrhage of the gastrointestinal mucosa, subendocardium, adrenal gland, and meninges; and edema of the gall bladder. Histologic examination confirmed the gross observations. Additional microscopic lesions included widespread degeneration and necrosis of the lymphoid tissues as well as of the surface and crypt epithelium of the gastrointestinal mucosa; mild scattered necrosis of pancreatic acinar cells, myocardium, bone marrow cells, adrenal cortical cells, and tubular epithelium of renal medulla; and mild interstitial pneumonia. A dose-dependent increase in lesion severity was observed except for the pancreatic lesion which was slightly more apparent in the pigs from the 0.6 mg/kg group. These findings indicate that T-2 toxin-induced lesions in the lymphoid tissues and gastrointestinal tract of pigs are similar to those of other species the pancreas and heart should be considered as additional target organs in the pig, and both rapidly dividing cells and those with little or no turnover are damaged by T-2 toxin.

Development of a reverse transcription multiplex real-time PCR for the detection and genotyping of classical swine fever virus

Abstract A reverse transcription multiplex real-time PCR (RT-MRT-PCR) was developed for rapid detection and genotyping of classical swine fever virus (CSFV). The universal primers and specific TaqMan probes for each of the three genotypes, genotypes 1, 2, and 3, were designed within the 3′-UTR of the CSFV. Non-CSFV swine virus and clinical samples from specific pathogen-free (SPF) pigs were both demonstrated to be CSFV-negative by RT-MRT-PCR. The diagnostic sensitivity of RT-MRT-PCR was determined to be 1 viral copy/μl for each genotype of standard plasmid. For the analytical sensitivity experiment, 100 samples of 14 CSFV genotype 1 strains and 86 samples from CSFV outbreak farms were all detected as CSFV-positive by RT-MRT-PCR, and the genotype results were consistent with the results of sequencing from a previous study. The intra-assay and inter-assay variations of RT-MRT-PCR were below 3% in all experiments. The sensitivity of RT-MRT-PCR was the same as the reverse transcription nested PCR (RT-nPCR) and higher than reverse transcription PCR (RT-PCR) and viral isolation from clinical samples. This assay was used further to evaluate the duration of viremia of wild-type CSFV in vaccinated exposed pigs. The results indicated that pigs vaccinated with the E2 subunit vaccine had longer viremia than pigs given the C-strain vaccine, which is compatible with the findings of previous studies. Thus, the new RT-MRT-PCR is a rapid, reproducible, sensitive, and specific genotyping tool for CSFV detection.

The immunogenicity of DNA constructs co-expressing GP5 and M proteins of porcine reproductive and respiratory syndrome virus conjugated by GPGP linker in pigs.

The heterodimer of glycoprotein 5 (GP5) and non-glycosylated matrix protein (M) is the leading target for the development of new generation of vaccines against porcine reproductive and respiratory syndrome virus (PRRSV) infection. It has been demonstrated that DNA vaccine co-expressing GP5 and M proteins as a fusion protein aroused better immunogenicity than that expressing GP5 or M alone, but it was no better than the DNA vaccine co-expressing GP5 and M proteins with two different promoters. Altered natural conformation of the co-expressed GP5 and M fusion protein was considered as the major cause. Glycine-proline-glycine-proline (GPGP) linker can minimize the conformational changes in tertiary structure and provide flexibility of the peptide chain. The objective of this study was to evaluate whether the immunogenicity of DNA constructs co-expressing GP5 and M proteins linked by GPGP could be enhanced in pigs. Three recombinant DNA constructs expressing GP5/M fusion protein without GPGP linker (pcDNA-56), GP5/M fusion protein conjugated by GPGP linker (pcDNA-5L6), and M/GP5 fusion protein conjugated by GPGP linker (pcDNA-6L5) were established. Sixteen PRRSV-free pigs were randomly assigned to four groups and inoculated intramuscularly with 3 consecutive doses of 500 μg of empty vector pcDNA3.1, pcDNA-56, pcDNA-5L6 or pcDNA-6L5 each at a 2-week interval followed by challenge with 5 × 10(5) TCID(50) PRRSV at 3 weeks after the final inoculation. All pcDNA-56-, pcDNA-5L6-, and pcDNA-6L5- but not pcDNA-3.1-inoculated pigs developed neutralizing antibodies (NAs) 3 weeks after the final inoculation and a gradual increase in NA titers after PRRSV challenge, indicating that pigs inoculated with these DNA constructs could establish a sufficient immune memory. The pcDNA-5L6- and pcDNA-6L5-inoculated pigs displayed lower level and shorter period of viremia and lower tissue viral load following PRRSV challenge than did the pcDNA-56-inoculated pigs. The strategy of co-expressing GPGP-linked GP5 and M fusion protein may be a promising approach for future PRRSV vaccine development, possibly via the improvement of natural conformation of the target fusion protein.