Development of a rabbit monocyte activation test as an alternative to the rabbit pyrogen test and its application in the analysis of plasma-derived products.

The rabbit pyrogen test (RPT) is a safety test conducted as a part of mandatory requirements of regulatory agencies. RPT is currently performed for routine quality control (QC) by manufacturers and for national lot release of biological products, such as plasma-derived products. However, RPT involves the use of many rabbits, counter to the international efforts to minimize the use of animals in research. Furthermore, pyrogen amount cannot be discerned from the test results and the results may be considerably affected by various factors. Therefore, a need exists for substituting RPT with in vitro assays. As a viable alternative to RPT, we here established a rabbit monocyte activation test (RMAT) based on the human MAT in the European Pharmacopoeia. RMAT uses rabbit peripheral blood mononuclear cells as the source of monocytes instead of live animals. The test detected endotoxin, lipoteichoic acid, peptidoglycan, and zymosan with high sensitivity, showing high correlation with the in vivo RPT results. The results of RMAT and RPT testing of non-pyrogenic plasma-derived products were also consistent. Furthermore, RMAT showed satisfactory recovery rates in an interference test with product samples and spiked-in pyrogens. We conclude that RMAT could replace the existing RPT for routine QC.

Comparison of porcine circovirus type 2 (PCV2)-associated lesions produced by co-infection between two genotypes of PCV2 and two genotypes of porcine reproductive and respiratory syndrome virus.

The objective of this study was to compare the virulence and pathogenicity of a combination of concurrent infections of two genotypes of porcine circovirus type 2 (PCV2) and two genotypes of porcine reproductive and respiratory syndrome virus (PRRSV) in terms of PCV2 viraemia, and PCV2-associated lesions and antigens in co-infected pigs. Pigs with PCV2a (or 2b)/type 1 (or type 2) PRRSV had significantly (P<0.05) higher mean clinical respiratory scores and lower average daily weight gain compared with pigs with PCV2a (or 2b). Co-infection induced significantly lower levels of anti-PCV2 and anti-PRRSV IgG antibodies than infection with one genotype alone, regardless of the genotype of the two viruses. Pigs with PCV2a (or 2b)/type 2 PRRSV had significantly (P<0.05) higher levels of PCV2 viraemia, more severe PCV2-associated lesions, and more PCV2 DNA within the lesions compared with pigs with PCV2a (or 2b)/type 1 PRRSV. However, there was no significant difference in these parameters in pigs with PCV2a/type 2 PRRSV or PCV2b/type 2 PRRSV. The results of this study demonstrate significant differences in the virulence and pathogenicity of type 1 and type 2 PRRSV but no significant differences in the virulence and pathogenicity of PCV2a and PCV2b with respect to the production of PCV2-associated lesions.

Vaccination of sows against type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) before artificial insemination protects against type 2 PRRSV challenge but does not protect against type 1 PRRSV challenge in late gestation.

The objective of the present study was to determine the effects of the commercially available type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)-based modified live vaccine against type 1 and type 2 PRRSV challenge in pregnant sows. Half of the sows in the study were vaccinated with a type 2 PRRSV-based vaccine 4 weeks prior to artificial insemination while the other half remained non-vaccinated. Sows were then challenged intranasally with type 1 or type 2 PRRSV at 93 days of gestation. The sows which received the type 2 PRRSV-based vaccine followed by type 2 PRRSV challenge had significantly higher neutralizing antibody titers against type 2 PRRSV than they did against type 1 PRRSV. These same sows had higher frequencies of IFN-γ-secreting cells when stimulated with type 2 PRRSV compared to those stimulated with type 1 PRRSV. Subsequent virological evaluation demonstrated that the type 2 PRRSV-based vaccine reduced the type 2 PRRSV load but not the type 1 PRRSV load present in the blood of the sows. Additionally, vaccination of pregnant sows with the type 2 PRRSV-based vaccine effectively reduced the level of type 2 PRRSV nucleic acids observed in fetal tissues from type 2 PRRSV-challenged sows but did not reduce the level of type 1 PRRSV nucleic acid observed in fetal tissues from type 1 PRRSV-challenged sows. This study demonstrates that the vaccination of pregnant sows with the type 2 PRRSV-based vaccine protects against type 2 PRRSV challenge but does not protect against type 1 PRRSV challenge.

Effect of porcine circovirus type 2 (PCV2) vaccination on PCV2-viremic piglets after experimental PCV2 challenge.

The objective of this study was to evaluate the effect of porcine circovirus type 2 (PCV2) vaccines on PCV2-viremic and -seropositive piglets born from naturally PCV2-infected sows against postnatal PCV2 challenge. The experimental design was aimed at mimicking commercial swine rearing conditions to evaluate the response of the PCV2 vaccine on PCV2-viremic and -seropositive piglets after experimental PCV2 challenge. PCV2a (or 2b)-viremic piglets received a PCV2 vaccine at 21 days of age followed by a PCV2b (or 2a) challenge at 49 days of age (28 days post vaccination). The PCV2 vaccines elicited a high level of humoral (as measured by immunoperoxidase monolayer assay and neutralizing antibody titers) and cellular (as measured by the frequency of PCV2-specific interferon-γ-secreting cells) immune response in the PCV2-viremic piglets after vaccination even in the presence of maternally derived antibodies (MDA). The initial infection of PCV2 in the pigs was not affected by PCV2 vaccination, however the challenging PCV2 was reduced by PCV2 vaccination on PCV2-viremic pigs. The results from this study demonstrate that the PCV2 vaccine used in this study is effective at reducing PCV2 viremia and lymphoid PCV2 DNA, even for PCV2-viremic pigs with passively acquired MDA at the time of vaccination.

Comparative pathogenesis of type 1 (European genotype) and type 2 (North American genotype) porcine reproductive and respiratory syndrome virus in infected boar.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) now has two main genotypes, genotype 1 (European) and genotype 2 (North American). There is a lack of data on the comparison of pathogenicity of the two genotypes in boars. The objectives of the present study were to evaluate the amount of PRRSV present in semen over time and compare the viral distribution and microscopic lesions of type 1 and type 2 PRRSV-infected boars. METHODS: Twenty-four 8-month-old PRRSV-naïve Duroc boars were randomly allocated to 3 treatment groups. The boars in groups 1 (n = 9) and 2 (n = 9) were intranasally inoculated with type 1 or type 2 PRRSV, respectively. The boars in groups 1 (n = 6) served as negative controls. Semen and blood samples were collected up to 35 days post-inoculation (dpi), and necropsies were performed on 14, 21, and 35 dpi. RESULTS: There were no significant differences in the genomic copy number of PRRSV, microscopic testicular lesion score, number of PRRSV-positive germ cells, or number of apoptotic cells between the type 1 and type 2 PRRSV-infected boars throughout the experiment. Histopathological changes were manifested by the desquamation of spermatocytes and the presence of multinucleated giant cells in seminiferous tubules of both type 1 and type 2 PRRSV-infected boars. The distribution of PRRSV-positive cells was focal; the virus was found in single germ cells or small clusters of germ cells, localized to the spermatogonia, spermatocytes, spermatids, and non-sperm cells in type 1 and type 2 PRRSV-infected boars. CONCLUSIONS: The results of this study demonstrated that two genotypes of PRRSV do not have significantly different virulence toward the male reproductive system of pigs.

GOLGA2/GM130, cis-Golgi matrix protein, is a novel target of anticancer gene therapy.

Achievement of long-term survival of patients with lung cancer treated with conventional chemotherapy is still difficult for treatment of metastatic and advanced tumors. Despite recent progress in investigational therapies, survival rates are still disappointingly low and novel adjuvant and systemic therapies are urgently needed. A recently elucidated secretory pathway is attracting considerable interest as a promising anticancer target. The cis-Golgi matrix protein, GOLGA2/GM130, plays an important role in glycosylation and transport of protein in the secretory pathway. In this study, the effects of short hairpin RNA (shRNA) constructs targeting GOLGA2/GM130 (shGOLGA2) on autophagy and lung cancer growth were evaluated in vitro and in vivo. Downregulation of GOLGA2/GM130 led to induction of autophagy and inhibition of glycosylation in A549 cells and in the lungs of K-ras(LA1) mice. Furthermore, downregulation of GOLGA2/GM130 decreased angiogenesis and cancer cell invasion in vitro and suppressed tumorigenesis in lung cancer mice model. The tumor specificity of sequence targeting GOLGA2/GM130 was also demonstrated. Taken together, these results suggest that induction of autophagy by shGOLGA2 may induce cell death rather than cell survival. Therefore, downregulation of GOLGA2/GM130 may be a potential therapeutic option for lung cancer.

Protective effect of the maternally derived porcine circovirus type 2 (PCV2)-specific cellular immune response in piglets by dam vaccination against PCV2 challenge.

The objective of the present study was to evaluate (i) the passive transfer of maternally derived functional porcine circovirus type 2 (PCV2)-specific lymphocytes of seronegative sows immunized with the PCV2 vaccine to newborn piglets and (ii) the functional role of the maternally derived PCV2-specific cellular immune response in protecting newborn piglets from challenge with PCV2. After ingesting colostrums, piglets from vaccinated sows (PT01 and PT02) have significantly higher numbers of PCV2-specific gamma interferon-secreting cells, an increased PCV2-specific delayed type hypersensitivity response, and a stronger proliferative response of peripheral blood mononuclear cells compared with piglets from non-vaccinated seronegative sows (PT03 and PT04). In the PCV2 challenge study, the number of serum genomic PCV2 copies was significantly less in piglets from vaccinated sows (PT02) compared with piglets from non-vaccinated sows (PT04) at 7-28 days post-inoculation (P<0.05 and P<0.001). The histopathological lesions and immunohistochemical scores were significantly lower in piglets of vaccinated sows compared with those of non-vaccinated sows. To our knowledge, this is the first report of transferring a maternally derived PCV2-specific cellular immune response from vaccinated dams to their offspring. Maternally derived adaptive cellular immune responses play a critical role in protecting newborn piglets challenged with PCV2 at 3 weeks of age.

Reduction of porcine circovirus type 2 (PCV2) viremia by a reformulated inactivated chimeric PCV1-2 vaccine-induced humoral and cellular immunity after experimental PCV2 challenge.

BACKGROUND: The objective of the present study was to elucidate the humoral and cellular immune response mechanisms by which a reformulated inactivated chimeric PCV1-2 vaccine reduces the PCV2 viremia. Forty PCV2 seronegative 3-week-old pigs were randomly divided into the following four groups: vaccinated challenged (T01), vaccinated non-challenged (T02), non-vaccinated challenged (T03), and non-vaccinated non-challenged (T04) animals. The pigs in groups T01 and T02 were immunized with a reformulated inactivated chimeric PCV1-2 vaccine (Fostera™ PCV; Pfizer Animal Health) administered as a 2.0 ml dose at 21 days of age. At 35 days of age (0 days post-challenge), the pigs in groups T01 and T03 were inoculated intranasally with 2 ml each of PCV2b. RESULTS: A reduction of PCV2 viremia coincided with the appearance of both PCV2-specific neutralizing antibodies (NA) and interferon-γ-secreting cells (IFN-γ-SCs) in the vaccinated animals. However, the presence of anti-PCV2 IgG antibodies did not correlate with the reduction of PCV2 viremia. Lymphocyte subset analysis indicated that the numbers of CD3+ and CD4+ cells increased in vaccinated animals but the numbers of CD4+ cells decreased transiently in non-vaccinated animals. The observation of a delayed type hypersensitivity response in only the vaccinated animals also supports a CD4+ cell-associated protective cellular immune response induced by the reformulated inactivated chimeric PCV1-2 vaccine. CONCLUSIONS: The induction of PCV2-specific NA and IFN-γ-SCs, and CD4+ cells by the reformulated inactivated chimeric PCV1-2 vaccine is the important protective immune response leading to reduction of the PCV2 viremia and control of the PCV2 infection. To our knowledge this is the first demonstration of protective humoral and cellular immunity induced by the reformulated inactivated chimeric PCV1-2 vaccine and its effect on reduction of PCV2 viremia by vaccination.

Chemoenzymatic synthesis of rivastigmine via dynamic kinetic resolution as a key step.

A practical and efficient procedure for the synthesis of rivastigmine was developed. This procedure includes dynamic kinetic resolution using a polymer-bound ruthenium complex and a lipase in combination as a key step. Enantiopure (-)-rivastigmine was obtained from commercially available 3'-hydroxyacetophenone via five steps in overall 57% yield.

Development of in situ hybridization assay that differentiates between two genotypes of Porcine circovirus-2 in formalin-fixed, paraffin-embedded tissues.

The aim of the current study was to develop a nonradioactive in situ hybridization assay that can differentiate between genotypes 2a and 2b of Porcine circovirus-2 (PCV-2) in formalin-fixed, paraffin-embedded lymph node tissues from pigs with postweaning multisystemic wasting syndrome. Two different digoxigenin-labeled oligonucleotide probes were designed from the PCV-2 open reading frame 2 sequences. The PCV-2a-specific probe did not hybridize with formalin-fixed, paraffin-embedded lymph nodes from naturally PCV-2b-infected pigs and vice versa. Both PCV-2a-specific and PCV-2b-specific probes gave consistent negative signals in lymph nodes from naturally PCV-1-infected pigs. The in situ hybridization assay described in the present study represents a diagnostic tool that can differentiate between the 2 genotypes of PCV-2.

Development of in situ hybridization for the detection of Mycoplasma hyorhinis in formalin-fixed paraffin-embedded tissues from naturally infected pigs with polyserositis.

The aim of this study was to develop in situ hybridization for detection of Mycoplasma hyorhinis in formalin-fixed, paraffin-wax-embedded tissues from pigs with polyserositis. M. hyorhinis was isolated from the spleen (2 pigs) and pericardium (1 pig). M. hyorhinis DNA was detected 16 out of 20 pigs with polyserositis. In situ hybridization produced a distinct positive signal for the M. hyorhinis p37 gene in inflammatory cells in the polyserositis. In situ hybridization developed in the present study present diagnostic tools capable of detection of M. hyorhinis in formalin-fixed, paraffin-wax-embedded tissues from the naturally infected pigs.

Standardization of the first Korean national reference standard for snake (Gloydius brevicaudus) antivenom.

In 2017, the second national reference standard (NRS) for Gloydius snake venom was established to replace the first NRS for Gloydius snake venom. In connection with the second venom NRS, a candidate for the first NRS for Gloydius snake antivenom was produced in 2017. In this study, the qualification of the candidate was estimated and the potency was determined by a collaborative study. The potency (anti-lethal titer and anti-hemorrhagic titer) of the candidate was determined by measuring the capability of the antivenom to neutralize the lethal and hemorrhagic effects of the second NRS for Gloydius snake venom, which was calibrated against the regional reference standard for Gloydius snake antivenom established in 2006. Two Korean facilities contributed data from 20 independent assays. Subsequently, one foreign national control research laboratories participated in this collaborative study. The general common potency of the anti-lethal and anti-hemorrhagic titers was obtained from the results of a total of 25 tests performed at three facilities. According to the results of the present study, the candidate preparation showed good quality and is judged to be suitable to serve as the first NRS for Gloydius snake antivenom with the following potency: an anti-lethal titer of 3100 unit (U) (95% confidence interval 2991-3276 U) and anti-hemorrhagic titer of 3000 U (95% confidence interval 2849-3159 U). In conclusion, the first NRS for Gloydius snake antivenom was established in this study. This reference standard will be used routinely for quality control of a snake antivenom product by manufacturer in Korea, which also can be used for national quality control, including a national lot-release test of the snake antivenom product.

Synthesis of optically active amino acid derivatives via dynamic kinetic resolution.

The complete conversion of racemic amino acid amides to optically active amino acid derivatives was accomplished via lipase/palladium-catalyzed dynamic kinetic resolution (DKR). In the DKR, a lipase catalyzes the selective acylation of L-substrate in the presence of acyl donor while unreacted D-substrate is isomerized by a Pd nanocatalyst to L-substrate. The DKR reactions provided good yields (80-98%) and high enantiomeric excess (95-98% ee). Interestingly, the DKR reactions of phenylglycine amide in the presence of Z-Gly-OMe or Z-Gly-Gly-OMe yielded optically active di- and tripeptide .

Comparative study of in situ hybridization and immunohistochemistry for the detection of porcine circovirus 2 in formalin-fixed, paraffin-embedded tissues.

In situ hybridization and immunohistochemistry with different types of antibody (monoclonal vs. polyclonal, natural vs. synthetic) was compared to detect porcine circovirus 2 (PCV2) in formalin-fixed, paraffin-embedded tissues from pigs with experimentally and naturally occurring postweaning multisystemic wasting syndrome. PCV2 DNA and antigen was detected in tissues from both experimentally and naturally infected pigs by in situ hybridization and immunohistochemistry, respectively. Statistical evaluation revealed that more PCV2 positive signals were significantly detected in both experimentally and naturally infected pigs by in situ hybridization compared with immunohistochemistry (P<0.05). The results of this study demonstrated that in situ hybridization proved more sensitive than immunohistochemistry for the detection of PCV2 in formalin-fixed, paraffin-embedded lymph node tissues.

Highly enantioselective dynamic kinetic resolution of 1,2-diarylethanols by a lipase-ruthenium couple.

A practical procedure has been developed for the dynamic kinetic resolution of 1,2-diarylethanols. This procedure employs a highly enantioselective lipase from Pseudomonas stutzeri (trade name, lipase TL) as the resolution catalyst and a ruthenium complex as the racemization catalyst. Sixteen 1,2-diarylethanols have been efficiently resolved to provide their acetyl derivatives with good yields (95-97%) and high enantiomeric excesses (96-99%).

A Collaborative Study to Establish the Second Korean National Reference Standard for Snake Venom.

In 2015, a candidate for the second national reference standard (NRS) of Gloydius snake venom was produced to replace the first NRS of Gloydius snake venom. In the present study, the potencies of the candidate were determined by a collaborative study, and the qualification of the candidate was estimated. The potencies of the candidate were determined by measuring the murine lethal titers and lapine hemorrhagic titers of venom against the regional working reference standard (RWRS) for antivenom using the methods described in the previous report for the first NRS of Gloydius snake venom. Three Korean facilities contributed data from a total of 30 independent assays. Subsequently, two foreign national control research laboratories contributed to this collaborative study. The results were calculated using the Reed-Muench method for lethality and determined using a mixed-effects model for hemorrhage. The general common potencies of the lethal and hemorrhagic titers were obtained from the results of the 30 tests performed at three Korean facilities. The results are expressed in micrograms for 1 test dose (TD) with a 95% confidence interval as follows: a lethal titer of 90.13 µg/TD (95% confidence interval = 87.39~92.86 µg) and a hemorrhagic titer of 10.80 µg/TD (95% confidence interval = 10.46~11.14 µg). In addition, the candidate preparation showed good quality evaluation according to the results of the quality estimation of the candidate and is judged to be suitable to serve as the Korean NRS for snake venom. In conclusion, the second NRS of Gloydius snake venom was established in this study and will be used for national quality control, including a national lot release test of Korean antivenom products.

Dynamic kinetic resolution of primary amines with a recyclable Pd nanocatalyst for racemization.

A practical procedure for the dynamic kinetic resolution (DKR) of primary amines has been developed. This procedure employs a palladium nanocatalyst as the racemization catalyst, a commercial lipase (Novozym-435) as the resolution catalyst, and ethyl acetate or ethyl methoxyacetate as the acyl donor. Eleven primary amines and one amino acid amide have been efficiently resolved with good yields (85-99%) and high enantiomeric excesses (97-99%). [reaction: see text]

Comparative analyses of humoral and cell-mediated immune responses upon vaccination with different commercially available single-dose porcine circovirus type 2 vaccines.

The objective of this study was to compare the induction of humoral and cell-mediated immune responses by four commercially available single-dose porcine circovirus type 2 (PCV-2) vaccines. A total of 50 3-week-old piglets were assigned to five groups (10 pigs per group). Four commercial PCV-2 vaccines were administered according to the manufacturer's instructions and the piglets were observed for 154 days post vaccination (dpv). Inactivated chimeric PCV-1-2 vaccines induced higher levels of PCV-2-specific neutralizing antibodies (NA) and interferon-γ-secreting cells (IFN-γ-SC) in pigs than did the other three commercial PCV-2 vaccines. The proportions of CD4(+) cells were significantly higher in animals vaccinated with inactivated chimeric PCV-1-2 and PCV-2 vaccines than in animals vaccinated with the two subunit vaccines. To our knowledge, this is the first comparison of humoral and cell-mediated immunity induced by four commercial single-dose PCV-2 vaccines under the same conditions. The results of this study demonstrated quantitative differences in the induction of humoral and cell-mediated immunity following vaccination.

Evaluation of the efficacy of a new modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine (Fostera PRRS) against heterologous PRRSV challenge.

The objective of this study was to evaluate a new modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine (Fostera PRRS, Zoetis, Florham, NJ, USA) that was based on a virulent US PRRSV isolate (P129) attenuated using CD163-expressing cell lines. Sixty-four PRRSV-seronegative 3-week-old pigs were randomly divided into the following four groups: vaccinated challenged (group 1), vaccinated unchallenged (group 2), unvaccinated challenged (group 3), and unvaccinated unchallenged (group 4). The pigs in groups 1 and 2 were immunized with a 2.0 mL dose of modified live PRRSV vaccine at 21 days of age, according to the manufacturer's recommendations. At 56 days of age (0 days post-challenge), the pigs in groups 1 and 3 were inoculated intranasally with 3 mL of tissue culture fluid containing 10(5) 50% tissue culture infective dose (TCID50)/mL of PRRSV (SNUVR090851 strain, fourth passage in MARC-145 cells). Vaccinated challenged pigs exhibited significantly lower (P<0.05) respiratory scores, viremia, macroscopic and microscopic lung lesion scores, and PRRSV-antigen with interstitial pneumonia than unvaccinated challenged pigs. The induction of PRRSV-specific IFN-γ-SCs by the new modified live PRRSV vaccine produced a protective immune response, leading to the reduction of PRRSV viremia. Although the new modified live PRRSV vaccine is not effective against heterologous PRRSV challenge, the new modified live PRRSV vaccine was able to reduce the levels of viremia and nasal shedding, and severity of PRRSV-induced lesions after challenging virus under experimental conditions.

Clinical, virological, immunological and pathological evaluation of four porcine circovirus type 2 vaccines.

The objective of this study was to rigorously compare the efficacy of four porcine circovirus type 2 (PCV2) vaccines of varying antigen type and dose under experimental conditions based on well-defined clinical (average daily weight gain [ADWG]), virological (evidence of viraemia), immunological (presence of PCV2-specific neutralising antibodies [NA], interferon-γ-secreting cells [IFN-γ-SCs], and CD3(+) and CD4(+) T cell subsets), and pathological (lymphoid lesion and PCV2 antigen score) criteria. A total of 60, 3-week old piglets were assigned to six groups of 10/group and were vaccinated either with 1/4 commercially available one-dose vaccines or were not vaccinated. At 7 weeks of age, vaccinated and control animals were inoculated intranasally with 2 mL of PCV2b. All pigs were euthanased and subjected to post-mortem examination at 25 weeks of age. From 9 to 16 weeks of age, the ADWG of vaccinated animals was significantly higher than that of non-vaccinates. Significant (P<0.05) differences were observed between vaccinated and positive control groups in the quantity of log-transformed PCV2b DNA in the blood and nasal swabs, log-transformed NA titres, and PCV2-specific IFN-γ-SCs at 0, 7, 14, 21, and 42 days post challenge (dpc). The proportion of CD4(+) cells at 7 and 14 dpc was also significantly different between vaccinated and control pigs (P<0.05). The histopathological lesions and PCV2-antigen scores in the lymph nodes were significantly lower (P<0.05) in vaccinated animals. All four vaccines were found to be highly efficacious in controlling experimental PCV2 challenge based on this range of criteria.