Porphyromonas gingivalis (Pg) is a keystone pathogen associated with chronic periodontitis and produces outer membrane vesicles (OMVs) that contain lipopolysaccharide (LPS), gingipains, and pathogen-derived DNA and RNA. Pg-OMVs are involved in the pathogenesis of periodontitis. Pg-OMV-activated pathways that induce the production of the pro-inflammatory cytokines, interleukin (IL)-6, and IL-8 in the human gingival epithelial cell line, OBA-9, were investigated. The role of mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB in levels of Pg-OMV-induced pro-inflammatory cytokines was investigated using Western blot analysis and specific pathway inhibitors. Pg-OMVs induced IL-6 and IL-8 production via the extracellular signal-regulated kinase (Erk) 1/2, c-Jun N-terminal kinase (JNK), p38 MAPK, and NF-κB signaling pathways in OBA-9 cells. In addition, the stimulator of interferon genes (STING), an essential innate immune signaling molecule, was triggered by a cytosolic pathogen DNA. Pg-OMV-induced IL-6 and IL-8 mRNA expression and production were significantly suppressed by STING-specific small interfering RNA. Taken together, these results demonstrated that Pg-OMV-activated Erk1/2, JNK, p38 MAPK, STING, and NF-κB signaling pathways resulting in increased IL-6 and IL-8 expression in human gingival epithelial cells. These results suggest that Pg-OMVs may play important roles in periodontitis exacerbation by stimulating various pathways.

Antigen-combining sites of the camelid heavy-chain antibody variable domain (VHH) are constructed by three complementarity-determining regions (CDR1, CDR2 and CDR3). We prepared cDNA using mRNA extracted from peripheral lymphocytes of alpacas that had been non-immunized or immunized with human serum albumin (HSA). The VHH gene fragments encoding the amino-terminal half-containing CDR1 as well as CDR2 and the carboxy-terminal half-containing CDR3 were amplified independently by PCR, and then full-length VHH gene fragments were generated by overlap extension PCR and cloned into the phagemid vector. This protocol, referred to as CDR shuffling, allowed us to construct an alpaca VHH phage display library possessing repertoires different from those naturally occurring in animals. We asked, first, whether this library was able to provide the functional VHH fragments against HSA, an immunized antigen, and obtained 29 anti-HSA VHH clones, 41% possessed K_D values of lower than 10⁻⁸ M, 5 of which had K_D values of 10⁻¹⁰ M. We also obtained VHH clones against non-immunized protein antigens such as cardiac troponin T and I, Ebola virus glycoprotein 1 and human immunoglobulin G by biopanning. We compared the amino acid sequences and affinities and found that 43% of VHHs had K_D values of less than 10⁻⁸ M, although those having K_D values of 10⁻¹⁰ M were unavailable. These results suggested that the CDR-shuffled VHH phage display library could potentially provide VHHs against non-immunized protein antigens with similar levels of affinities to those against immunized antigens.

A new alpaca VHH antibody library against 3-(1-naphthoyl)-indole derivatives was developed from alpaca immunized with 7-(3-(1-naphthoyl)-1H-indol-1-yl)-heptanoic acid-keyhole limpet hemocyanin (Hep-KLH) protein conjugates as the immunogen. From this library, two 3-(1-naphthoyl)-indole derivative-specific clones, named NN01 and NN02, were isolated using biopanning technology. The binding specificity of these clones was confirmed using a competitive enzyme-linked immunosorbent assay (c-ELISA). Based on the results of c-ELISA, a median inhibitory concentration (IC ₅₀ ) of these two VHH antibodies, NN01 and NN02, in the case of 7-(3-(1-naphthoyl)-1H-indol-1-yl)-heptanoic acid (Hep; one of 3-(1-naphthoyl)-indole derivatives) as an inhibitor exhibited an approximate 3 × 10 ⁻⁷ M and 6 × 10 ⁻⁷ M, respectively. Thus, VHH antibodies produced in this study could be considered a useful tool for the detection of 3-(1-naphthoyl)-indole derivatives.

Laryngeal papilloma (LP), which is associated with infection by human papillomavirus (HPV)-6 or -11, displays aggressive growth. The precise molecular mechanism underlying the tumorigenesis of LP has yet to be uncovered. Building on our earlier research into HPV-6, in this study, the viral gene expression of HPV-11 was investigated by quantitative PCR and DNA/RNA in situ hybridization. Additionally, newly developed antibodies against the E4 protein of HPV-6 and HPV-11 were evaluated by immunohistochemistry. The average viral load of HPV-11 in LP was 1.95 ± 0.66 × 10⁵ copies/ng DNA, and 88% of HPV mRNA expression was found to be E4, E5a, and E5b mRNAs. According to RNA in situ hybridization, E4 and E5b mRNAs were expressed from the middle to upper part of the epithelium. E4 immunohistochemistry revealed a wide positive reaction in the upper cell layer in line with E4 mRNA expression. Other head and neck lesions with HPV-11 infection also showed a positive reaction in E4 immunohistochemistry. The distribution pattern of HPV DNA, viral mRNA, and E4 protein in LP with HPV-11 infection was quite similar to that of HPV-6. Therefore, it might be possible to apply these E4-specific antibodies in other functional studies as well as clinical applications, including targeted molecular therapies in patients with HPV-6 and HPV-11 infection.

Fibroblast growth factor (FGF) is a multifunctional protein that exhibits a wide range of biological effects. Most commonly, it acts as a mitogen, but it also has regulatory, morphological, and endocrine effects. The four receptor subtypes of FGF are activated by more than 20 different FGF ligands. FGF2, one of the FGF ligands, is an essential factor for cell culture in stem cells for regenerative medicine; however, recombinant FGF2 is extremely unstable. Here, we successfully generated homobivalent agonistic single domain antibodies (variable domain of heavy chain of heavy chain antibodies referred to as VHHs) that bind to the domain III and induce activation of the FGF receptor 1 (FGFR1) and thus transduce intracellular signaling. This agonistic VHH has similar biological activity (EC₅₀) as the natural FGF2 ligand. Furthermore, we determined that the agonistic VHH could support the proliferation of human induced pluripotent stem cells (hiPSCs) and human mesenchymal stem cells (hMSCs), which are pluripotent stem cells for regenerative medicine. Additionally, the agonistic VHH could maintain the ability of MCS cells to differentiate into adipocytes or osteocytes, indicating that it could maintain the properties of pluripotent stem cells. These results suggest that the VHH agonist may function as an FGF2 mimetic in cell preparation of stem cells for regenerative medicine with better cost effectiveness.

IgM antibodies (Abs) are thought to play a major role in humoral immunity but only at the early stage of the primary immune response. However, two subsets of IgM⁺ memory B cells (MBCs), one with high affinity gained by means of multiple somatic hypermutation (SHM) and the other with low affinity and no SHMs, are generated through the germinal center (GC)-dependent and GC-independent (non-GC) pathway, respectively, after immunization with (4-hydroxy-3-nitrophenyl)acetyl (NP)-chicken γ-globulin. Surprisingly, an analysis of antibody-secreting cells reveals that a large amount of anti-NP IgM Ab with few SHMs is secreted during the recall response, indicating that only non-GC MBCs have terminal differentiation potential. Since secondary IgM Abs are capable of binding to dinitrophenyl ligands, they likely provide broad cross-reactivity in defense against microbial infection.