PEGylation of protein and peptide drugs is frequently used to improve in vivo efficacy. We investigated the action mechanism of tachyplesin I, a membrane-acting cyclic antimicrobial peptide from Tachypleus tridentatus and the effects of PEGylation on the mechanism. The PEGylated peptide induced the leakage of calcein from egg yolk L-alpha-phosphatidylglycerol/egg yolk L-alpha-phosphatidylcholine large unilamellar vesicles similarly to the parent peptide. Both peptides induced lipid flip-flop coupled to leakage and was translocated into the inner leaflet of the bilayer, indicating that tachyplesin I forms a toroidal pore and that PEGylation did not alter the basic mechanism of membrane permeabilization of the parent peptide. Despite their similar activities against model membranes, the peptides showed very different biological activities. The cytotoxicity of tachyplesin I was greatly reduced by PEGylation, although the antimicrobial activity was significantly weakened. We investigated the enhancement of the permeability of inner membranes induced by the peptides. Our results suggested that outer membranes and peptidoglycan layers play an inhibitory role in the permeation of the PEG moiety. Furthermore, a reduction in DNA binding by PEGylation may also contribute to the weak activity of the PEGylated peptide.

Previously, we showed that bacterial DNA and vertebrate DNA/cationic liposome complexes stimulate potent inflammatory responses in cultured mouse macrophages. In the present study, we examined whether endocytosis and subsequent acidification are associated with these responses. The endocytosis inhibitor, cytochalasin B, reduced tumor necrosis factor ␣ (TNF-␣) production by a plasmid DNA (pDNA)/cationic liposome complex. The endosomal acidification inhibitor, monensin, inhibited cytokine production by pDNA or a calf thymus DNA/liposome complex. These results suggest, similarly to CpG motif-dependent responses, that endocytosis and subsequent endosomal acidification are also required for these inflammatory responses. It is intriguing that another inhibitor of endosomal acidification, bafilomycin A, stimulated the production of TNF-␣ mRNA and its protein after removal of the pDNA/liposome complex and inhibitors, although it inhibited the release of interleukin-6. Similar phenomena were observed in the activation of macrophages by CpG oligodeoxynucleotide, calf thymus DNA, and Escherichia coli DNA complexed with liposomes. Moreover, bafilomycin A also induced a high degree of TNF-␣ release after stimulation with naked pDNA. These results suggest that bafilomycin A increases TNF-␣ production induced by DNA at the transcriptional level via an as-yet unknown mechanism. Furthermore, we investigated the contribution of Toll-like receptor 9 (TLR9), the receptor of CpG motifs, to the cell activation by the DNA/cationic liposome complex using the macrophages from TLR9 ؊/؊ mice. We observed a reduced inflammatory cytokine release from macrophages of TLR9 ؊/؊ mice compared with wild-type mice. However, the cytokine production was not completely abolished, suggesting that the DNA/cationic liposome complex can induce macrophage activation via TLR9-dependent and -independent pathways. J. Leukoc. Biol. 77: 71-79; 2005.

No abstract

Acquired hemophilia A (AHA), which is caused by autoantibodies against coagulation factor VIII (FVIII) is a rare, life-threatening bleeding disorder, the incidence of which appears to be increasing in Japan as the population ages. However, the clinical characteristics, treatment, and outcomes of AHA remain difficult to establish due to the rarity of this disease. We retrospectively analyzed data from 25 patients (median age 73 years; range 24-92 years; male n = 15) diagnosed with AHA between 1999 and 2015 at Gunma University Hospital. We identified autoimmune diseases and malignancy as underlying conditions in four and three patients, respectively. Factor VIII activity was significantly decreased in all patients (median 2.0%; range <1.0-8.0) by FVIII inhibitor (median 47.0 BU/mL; range 2.0-1010). Among 71 bleeding events, subcutaneous or intramuscular hemorrhage was the most prevalent. Seventeen patients required bypassing agents. Twenty-two (91.7%) of 24 patients treated with immunosuppressive agents achieved complete response (CR) during a median of 57.5 days (range 19-714 days). Although three patients (12%) relapsed and seven (28%) died of infection, none of the deaths were related to bleeding. Although most of our patients achieved CR after immunosuppressive therapy, the rate of infection-related mortality was unsatisfactorily high.

SUMMARYThe production of in¯ammatory cytokines from macrophages (Mf), upon stimulation with plasmid DNA (pDNA) containing CpG motifs, is a critical process for DNA-based therapies such as DNA vaccination and gene therapy. We compared Mf activation, following stimulation with naked pDNA, based on the production of cytokines from cell lines (RAW264.7 and J774A1) and peritoneal Mfs in primary culture. The Mf cell lines RAW264.7 and J774A1 produced a signi®cant amount of tumour necrosis factor-a (TNF-a) upon stimulation with naked pDNA and this response required endosomal acidi®cation. On the other hand, peritoneal Mfs (both resident and elicited) in primary culture did not secrete TNF-a or interleukin-6, although they contain the mRNA of toll-like receptor-9 (TLR-9) and are able to respond to CpG oligodeoxynucleotides. This unresponsiveness was not a result of impaired cellular uptake of pDNA because the primary cultured Mfs showed a higher uptake of pDNA than the RAW264.7 and J774A1 cell lines. These ®ndings have important implications for Mf activation by naked pDNA as it has been generally assumed that pDNA that contains CpG motifs is a potent agent for inducing in¯ammatory cytokines in vivo, based on evidence from in vitro studies using Mf cell lines.

Human polyomavirus JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy, is ubiquitous in humans, infecting children asymptomatically then persisting in renal tissue. Since JCV DNA can readily be detected from urine, it should be a useful tool with which to study the mode of virus transmission in humans. Based on this notion, we examined the extent to which JCV was transmitted from the American to Japanese populations in Okinawa Island, Japan. (A population of about 50 000 American soldiers and families have been stationed in Okinawa since 1945.) Four JCV types (A to D) were identified in American populations in U.S.A., whereas only type B was prevalent in elder Japanese in Okinawa who had reached adulthood by 1945. Thus, types A, C, and D served as indicators of the transmission of JCV from American to Japanese populations. We then examined whether types A, C, and D were detectable in Japanese in Okinawa aged 30-50 years who may have been in contact with Americans during childhood. However, all the 125 isolates from the younger Japanese population were type B without exception. From this finding, we concluded that JCV is rarely transmitted between human populations.

No abstract

We demonstrated that the number of circulating pDCs is low in patients with primary and H. pylori-associated ITP and that it changes depending on treatment modality. Further investigation is warranted with regard to the role of pDCs in the immunopathogenesis of ITP.