Generation of Neutralizing Human Monoclonal Antibodies against Parvovirus B19 Proteins

Infections caused by human parvovirus B19 are known to be controlled mainly by neutralizing antibodies. To analyze the immune reaction against parvovirus B19 proteins, four cell lines secreting human immunoglobulin G monoclonal antibodies (MAbs) were generated from two healthy donors and one human immunodeficiency virus type 1-seropositive individual with high serum titers against parvovirus. One MAb is specific for nonstructural protein NS1 (MAb 1424), two MAbs are specific for the unique region of minor capsid protein VP1 (MAbs 1418-1 and 1418-16), and one MAb is directed to major capsid protein VP2 (MAb 860-55D). Two MAbs, 1418-1 and 1418-16, which were generated from the same individual have identity in the cDNA sequences encoding the variable domains, with the exception of four base pairs resulting in only one amino acid change in the light chain. The NS1- and VP1-specific MAbs interact with linear epitopes, whereas the recognized epitope in VP2 is conformational. The MAbs specific for the structural proteins display strong virus-neutralizing activity. The VP1- and VP2-specific MAbs have the capacity to neutralize 50% of infectious parvovirus B19 in vitro at 0.08 and 0.73 μg/ml, respectively, demonstrating the importance of such antibodies in the clearance of B19 viremia. The NS1-specific MAb mediated weak neutralizing activity and required 47.7 μg/ml for 50% neutralization. The human MAbs with potent neutralizing activity could be used for immunotherapy of chronically B19 virus-infected individuals and acutely infected pregnant women. Furthermore, the knowledge gained regarding epitopes which induce strongly neutralizing antibodies may be important for vaccine development.     

Preventing Staphylococcus aureus sepsis through the inhibition of its agglutination in blood

Staphylococcus aureus infection is a frequent cause of sepsis in humans, a disease associated with high mortality and without specific intervention. When suspended in human or animal plasma, staphylococci are known to agglutinate, however the bacterial factors responsible for agglutination and their possible contribution to disease pathogenesis have not yet been revealed. Using a mouse model for S. aureus sepsis, we report here that staphylococcal agglutination in blood was associated with a lethal outcome of this disease. Three secreted products of staphylococci--coagulase (Coa), von Willebrand factor binding protein (vWbp) and clumping factor (ClfA)--were required for agglutination. Coa and vWbp activate prothrombin to cleave fibrinogen, whereas ClfA allowed staphylococci to associate with the resulting fibrin cables. All three virulence genes promoted the formation of thromboembolic lesions in heart tissues. S. aureus agglutination could be disrupted and the lethal outcome of sepsis could be prevented by combining dabigatran-etexilate treatment, which blocked Coa and vWbp activity, with antibodies specific for ClfA. Together these results suggest that the combined administration of direct thrombin inhibitors and ClfA-antibodies that block S. aureus agglutination with fibrin may be useful for the prevention of staphylococcal sepsis in humans.     

Comparison of PERV genomic locations between Asian and European pigs

Xenotransplantation from pigs provides a possible solution to the shortage of human organs for allotransplantation. Porcine endogenous retroviruses (PERVs) are a possible obstacle to using porcine organs in addition to the immunological barriers. Three main types of PERVs (A, B and C) have been previously investigated in diverse pig breeds. To examine the copy numbers of PERVs and their genomic locations in the Korean native pig genome, we screened a BAC (Bacterial Artificial Chromosome) library with PERV-specific protease primers for initial recognition of PERV-positive clones and three sets of envelope-specific primers for the identification of PERV types. A total of 45 PERV-positive clones, nine PERV-A and 36 PERV-B, have been identified from the library screening and the BAC contigs were constructed using the primers designed from BAC end sequences (BESs). These primers were also used for SCH (Somatic Cell Hybrid) and RH (Radiation Hybrid) mapping of the PERV-positive clones. The results indicate that 45 PERV-positive BAC clones belong to nine contigs and a singleton. SCH and IMpRH (INRA-Minnesota Porcine Radiation Hybrid) mapping results indicated that there are at least eight separate PERV genomic locations, consisting of three PERV-A and five PERV-B. One contig could not be mapped, and two contigs are closely located on SSC7. Southern blotting indicates there may be up to 15 additional sites. Further investigation of these clones will contribute to a general strategy to generate PERV-free lines of pigs suitable for xenotransplantation.     

From proteomics toward systems biology: integration of different types of proteomics data into network models

Living organisms are comprised of various systems at different levels, i.e., organs, tissues, and cells. Each system carries out its diverse functions in response to environmental and genetic perturbations, by utilizing biological networks, in which nodal components, such as, DNA, mRNAs, proteins, and metabolites, closely interact with each other. Systems biology investigates such systems by producing comprehensive global data that represent different levels of biological information, i.e., at the DNA, mRNA, protein, or metabolite levels, and by integrating this data into network models that generate coherent hypotheses for given biological situations. This review presents a systems biology framework, called the 'Integrative Proteomics Data Analysis Pipeline' (IPDAP), which generates mechanistic hypotheses from network models reconstructed by integrating diverse types of proteomic data generated by mass spectrometry-based proteomic analyses. The devised framework includes a serial set of computational and network analysis tools. Here, we demonstrate its functionalities by applying these tools to several conceptual examples.     

Semi-continuous photo-fermentative H2 production by Rhodobacter sphaeroides: effect of decanting volume ratio

In this study, a semi-continuous operation of photo-fermentative H₂-producing reactor was attempted at various decanting volume ratios (DVR, decanting volume per day/total working volume, %), ranging 30-70%, using Rhodobacter sphaeroides KD131. H₂ production was not efficient with showing low H₂ yields of 0.2 and 0.5 mol H₂/mol succinate_added at 30% and 40% DVR, respectively. The low performance ascribed to the fact that over 70% of substrate electrons were diverted towards cell growth under these conditions. Meanwhile, cell growth was limited at DVR ? 50%; therefore, higher H₂ yields (>2.0 mol H₂/mol succinate_added) were observed. Both the highest H₂ yield of 3.7 mol H₂/mol succinate_added and production rate of 1494 mL H₂/L-reactor/d were achieved at 60% DVR. The content of soluble microbial products (SMPs) was measured, which accounted for 3-15% of substrate electrons. It was found that the largest (65-75%) portion of SMPs comprised low molecular-weight (<3 kDa).     

High‐density areas for harbor porpoises (Phocoena phocoena) identified by satellite tracking

The population status of harbor porpoises has been of concern for several years, and the establishment of Marine Protected Areas (MPAs) has been suggested as a method to protect the harbor porpoise (Phocoena phocoena, Linneaus 1758) and other small cetaceans. In order to designate MPAs, high‐density areas for the species must be identified. Spatial distribution of small cetaceans is usually assessed from ship or aerial surveys. As a potentially more accurate alternative, this study examined the movements and area preferences of 64 harbor porpoises, satellite tagged between 1997 and 2007, in order to determine the distribution in the North Sea, the western Baltic, and the waters in between. Results show that harbor porpoises are not evenly distributed, but congregate in nine high‐density areas within the study area. Several of these areas are subject to significant seasonal variation. The study found no differences in the home range size of males and females, but immature harbor porpoises have larger home ranges than mature porpoises. The use of satellite telemetry for identifying areas of high harbor porpoise density can be of key importance when designating MPAs.     

Retigeric acid B exhibits antitumor activity through suppression of nuclear factor-κB signaling in prostate cancer cells in vitro and in vivo

Previously, we reported that retigeric acid B (RB), a natural pentacyclic triterpenic acid isolated from lichen, inhibited cell growth and induced apoptosis in androgen-independent prostate cancer (PCa) cells. However, the mechanism of action of RB remains unclear. In this study, we found that using PC3 and DU145 cells as models, RB inhibited phosphorylation levels of IκBα and p65 subunit of NF-κB in a time- and dosage-dependent manner. Detailed study revealed that RB blocked the nuclear translocation of p65 and its DNA binding activity, which correlated with suppression of NF-κB-regulated proteins including Bcl-2, Bcl-x(L), cyclin D1 and survivin. NF-κB reporter assay suggested that RB was able to inhibit both constitutive activated-NF-κB and LPS (lipopolysaccharide)-induced activation of NF-κB. Overexpression of RelA/p65 rescued RB-induced cell death, while knockdown of RelA/p65 significantly promoted RB-mediated inhibitory effect on cell proliferation, suggesting the crucial involvement of NF-κB pathway in this event. We further analyzed antitumor activity of RB in in vivo study. In C57BL/6 mice carrying RM-1 homografts, RB inhibited tumor growth and triggered apoptosis mainly through suppressing NF-κB activity in tumor tissues. Additionally, DNA microarray data revealed global changes in the gene expression associated with cell proliferation, apoptosis, invasion and metastasis in response to RB treatment. Therefore, our findings suggested that RB exerted its anti-tumor effect by targeting the NF-κB pathway in PCa cells, and this could be a general mechanism for the anti-tumor effect of RB in other types of cancers as well.     

Production of nattokinase by high cell density fed-batch culture of Bacillus subtilis

Bacillus subtilis was cultivated to high cell density for nattokinase production by pH-stat fed-batch culture. A concentrated mixture solution of glucose and peptone was automatically added by acid-supplying pump when culture pH rose above high limit. Effect of the ratio of glucose to peptone in feeding solution was investigated on cell growth and nattokinase production by changing the ratio from 0.2 to 5 g glucose/g peptone. The highest cell concentration was 77 g/L when the ratio was 0.2 g glucose/g peptone. Cell concentration decreased with increasing the ratio of glucose to peptone in feeding solution, while the optimum condition existed for nattokinase production. The highest nattokinase activity was 14,500 unit/mL at a ratio of 0.33 g glucose/g peptone, which was 4.3 times higher than that in batch culture.     

An intracellular antibody can suppress tumorigenicity in Hepatitis B virus X-expressing cells

Although the hepatitis B virus X protein (HBx) is thought to play a causative role in the development of hepatocellular carcinoma, it is not yet known whether interfering with HBx function may affect the cellular transformation of HBx-expressing tumor cells. To address this question, we adopted an intracellular antibody fragment expression approach to block the function of HBx. Expression of a single-chain variable fragment (scFv) specific to HBx (designated as H7scFv) inhibited HBx-dependent cellular transactivation. Furthermore, H7scFv suppressed the growth of HBx-expressing tumor cells in both soft agar and nude mice. The suppressive effect of H7scFv on tumorigenicity appeared not to be mediated by inhibition of HBx-induced growth stimulation since the growth rate of these cells was not affected significantly by H7scFv expression. In conclusion, these data suggest that the HBx-dependent transformed phenotype is reversible and that HBx may be a good molecular target for the treatment of HBV-related tumors.This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health& Welfare, Republic of Korea (03-PJ1-PG3-20200–0023)