Hematopoiesis-dependent expression of CD44 in murine hepatic progenitor cells

The fetal liver serves as the predominant hematopoietic organ until birth. However, the mechanisms underlying this link between hematopoiesis and hepatogenesis are unclear. Previously, we reported the isolation of a monoclonal antibody (anti-Liv8) that specifically recognizes an antigen (Liv8) present in murine fetal livers at embryonic day 11.5 (E11.5). Liv8 is a cell surface molecule expressed by hematopoietic cells in both fetal liver and adult mouse bone marrow. Here, we report that Liv8 is also transiently expressed by hepatoblasts at E11.5. Using protein purification and mass spectrometry, we have identified Liv8 as the CD44 protein. Interestingly, the expression of Liv8/CD44 in fetal liver was completely lost in AML1^−/− murine embryos, which lack definitive hematopoiesis. These results show that hepatoblasts change from Liv8/CD44-negative to Liv8/CD44-positive status in a hematopoiesis-dependent manner by E11.5, and indicate that Liv8/CD44 expression is an important link between hematopoiesis and hepatogenesis during fetal liver development.     

Detection of circulating Asian H5N1 viruses by a newly established monoclonal antibody

Monoclonal antibodies (MAbs) against the recently emerged Asian H5N1 virus (A/crow/Kyoto/53/2004) were generated. From five anti-hemagglutinin (HA) MAbs, four antibodies (3C11, 4C12, 3H12, and 3H4) broadly in vitro recognized and neutralized H5 subtypes, including H5N1. By contrast, the 4G6 MAb specifically reacted with H5N1-HA and not with H5N2- or H5N3-HAs from previous epidemics. The 4G6 MAb was useful for immunofluorescence assays but not for immunoblotting, suggesting that this antibody recognizes a conformational epitope of the H5N1-HA protein. An intensive epitope-mapping analysis demonstrated that the 4G6 MAb recognizes Asp59, which is highly conserved among currently circulating H5N1 lineages. Further, a 4G6-based antigen capture enzyme-linked immunosorbent assay detected H5N1 even that derived from clade 2.2 (A/chicken/Egypt/CL-61/2007) from infected chicken lung before virus isolation. Taken together, these results suggest that the established MAbs, especially 4G6, are useful for rapid and specific detection of Asian H5N1 viruses.     

Epitope Mapping of a Bactericidal Monoclonal Antibody against the Factor H Binding Protein of Neisseria meningitidis

The factor H binding protein (fHbp) is a 27-kDa membrane-anchored lipoprotein of Neisseria meningitidis that allows the survival of the bacterium in human plasma; it is also a major component of a universal vaccine against meningococcus B.In this study, we used nuclear magnetic resonance spectroscopy, mutagenesis, and in silico modeling to map the epitope recognized by MAb502, a bactericidal monoclonal antibody elicited by fHbp. The data show that the antibody recognizes a conformational epitope within a well-defined area of the immunodominant C-terminal domain of the protein that is formed by two loops connecting different β-strands of a β-barrel and a short α-helix brought in spatial proximity by the protein folding. The identification of the protective epitopes of fHbp is an important factor for understanding the mechanism(s) of an effective immune response and provides valuable guidelines for designing variants of the protein able to induce broadly protective immunity.     

Antigenemia and Specific IgM and IgG Antibody Responses in Rabbits Infected with Toxoplasma gondii

In this experiment, the correlation between antigenemia and specific antibody responses in Toxoplasma gondii-infected rabbits was assessed. We injected 1,000 T. gondii tachyzoites (RH) subcutaneously into 5 rabbits. Parasitemia, circulating antigens, and IgM and IgG antibody titers in blood were tested by ELISA and immunoblot. For detection of parasitemia, mice were injected with blood from rabbits infected with T. gondii and mice died between days 2 and 10 post-infection (PI). Circulating antigens were detected early on day 2 PI, and the titers increased from day 4 PI and peaked on day 12 PI. Anti-Toxoplasma IgM antibody titers increased on day 6 PI and peaked on days 14-16 PI. IgG was detected from day 10 PI, and the titers increased continuously during the experiment. The antigenic protein patterns differed during the infection period, and the number of bands increased with ongoing infection by the immunoblot analysis. These result indicated that Toxoplasma circulating antigens during acute toxoplasmosis are closely related to the presence of parasites in blood. Also, the circulating antigen levels were closely correlated with IgM titers, but not with IgG titers. Therefore, co-detection of circulating antigens with IgM antibodies may improve the reliability of the diagnosis of acute toxoplasmosis.     

Nippostrongylus brasiliensis: Increase of sialomucins reacting with anti-mucin monoclonal antibody HCM31 in rat small intestinal mucosa with primary infection and reinfection

Infections with the parasitic helminth, Nippostrongylus brasiliensis, cause changes in rat small intestinal goblet cell mucin, particularly in the peripheral sugar residues of oligosaccharide. These changes may correlate with expulsion. In this study, we examined changes in mucin oligosaccharides caused by primary infection and reinfection with N. brasiliensis, using two monoclonal antibodies, HCM31 and PGM34, that react with sialomucin and sulfomucin, respectively. Enzyme-linked immunosorbent assay of jejunal mucins showed that the relative reactivity of mucins with HCM31, but not PGM34, increased up to 16 days after primary infection and 6 days after reinfection, the times when the worms were expelled from the rats. Immunohistochemical studies confirmed that goblet cells stained with HCM31 greatly increased at the time of worm expulsion. These results indicate that the marked increase observed in HCM31-reactive sialomucins may be related to expulsion of the worms.     

Factor VIII-hydrolyzing IgG in acquired and congenital hemophilia

Anti-factor VIII (FVIII) inhibitory IgG may arise as alloantibodies to therapeutic FVIII in patients with congenital hemophilia A, or as autoantibodies to endogenous FVIII in individuals with acquired hemophilia. We have described FVIII-hydrolyzing IgG both in hemophilia A patients with anti-FVIII IgG and in acquired hemophilia patients. Here, we compared the properties of proteolytic auto- and allo-antibodies. Rates of FVIII hydrolysis differed significantly between the two groups of antibodies. Proline-phenylalanine-arginine-methylcoumarinamide was a surrogate substrate for FVIII-hydrolyzing autoantibodies. Our data suggest that populations of proteolytic anti-FVIII IgG in acquired hemophilia patients are different from that of inhibitor-positive hemophilia A patients.     

A new approach to monitor expression of aldo–keto reductase proteins in mouse tissues

The aldo–keto reductase (AKR) proteins catalyze reduction of diverse aldehydes and play detoxification roles in many organisms. Since many substrates are shared among AKR, it is generally accepted that these enzymes can functionally compensate each other in response to oxidative stress. Their overall abundances are the important factor that partially reflects the capacity of antioxidant and detoxification in tissues. In this study, the strategy was proposed for generation of Pan‐AKR antibodies to recognize most AKR proteins in mouse tissues. Derived from bioinformatic analysis, several consensus peptides with different potential antigenicities were synthesized, conjugated to hemocyanin from keyhole limpets and further delivered to rabbits to generate polyclonal antibodies. Three Pan‐AKR antibodies exhibited the immune specificities and immune sensitivities, Pan‐AKR‐P1 for AKR1B and AKR1C, Pan‐AKR‐P3 for AKR1C and Pan‐AKR‐P4 for all the AKR proteins. Pan‐AKR‐P4 antibody was employed to 2‐DE Western blot to examine the AKR abundances in mouse liver and kidney, resulting in seven immune‐reactive spots from each tissue. Protein identification with MS revealed that most immune‐positive spots were the members of AKR superfamily. Furthermore, Pan‐AKR‐P4 antibody was implemented to compare the different abundances of the AKR proteins in liver and kidney between normal and diabetic mice, suggesting that diabetes did cause some abnormal changes in the AKR protein abundances.     

Tandem multimer expression of angiotensin I-converting enzyme inhibitory peptide in Escherichia coli

It is common for small tandem peptide multimer genes to be indirectly inserted into expression vectors and fused with a protein tag. In this study, a multimer of the tandem angiotensin I-converting enzyme inhibitory peptide (ACE-IP) gene was directly transferred to a commercially available vector and the designed gene was expressed as a repeated peptide in Escherichia coli BL21(DE3)pLysS. The process further developed in our study was the construction of six-repeated ACE-IP synthetic genes and their direct insertion. Protein expression in inclusion bodies was confirmed by SDS-PAGE and Western blot. Acid hydrolysis of inclusion bodies produced single-unit peptides through cleavage of the aspartyl-prolyl bonds. This cleaved recombinant peptide (rACE-IP) was purified using immuno-affinity chromatography followed by reversed phase-HPLC. 105–115 mg of the lyophilized recombinant peptide was obtained from 1 L E. coli culture. In vitro biological activity of rACE-IP was indistinguishable from that of the natural peptide produced by hydrolysis in artificial gastric juice or by acidic hydrolysis. The rACE-IP prepared by recombinant DNA technology and solid-phase synthesis methods showed a similar IC₅₀. This strategy could be used for the expression of important peptides, which have N-terminal proline (P) and C-terminal aspartic acid residues (D) for commercial applications, e.g. functional foods and drinks.     

Application of a PEG/salt aqueous two-phase partition system for the recovery of monoclonal antibodies from unclarified transgenic tobacco extract

Aqueous two-phase partition systems (ATPS) have been widely used for the separation of a large variety of biomolecules. In the present report, the application of a polyethylene glycol/phosphate (PEG/phosphate) ATPS for the separation of anti-HIV monoclonal antibodies 2G12 (mAb 2G12) and 4E10 (mAb 4E10) from unclarified transgenic tobacco crude extract was investigated. Optimal conditions that favor opposite phase partitioning of plant debris/mAb as well as high recovery and purification were found to be 13.1% w/w (PEG 1500), 12.5% w/w (phosphate) at pH 5 with a phase ratio of 1.3 and 8.25% w/w unclarified tobacco extract load. Under these conditions, mAb 2G12 and mAb 4E10 were partitioned at the bottom phosphate phase with 85 and 84% yield and 2.4- and 2.1-fold purification, respectively. The proposed ATPS was successfully integrated in an affinity-based purification protocol, using Protein A, yielding antibodies of high purity and yield. In this study, ATPS was shown to be suitable for initial protein recovery and partial purification of mAb from unclarified transgenic tobacco crude extract.