The lipopeptide FSL-1 [S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe, Pam 2 CG DPKHPKSF] synthesized on the basis of the N-terminal structure of a Mycoplasma salivarium lipoprotein capable of activating normal human gingival fibroblasts to induce the cell surface expression of ICAM-1 revealed an activity to induce production of monocyte chemoattractant protein 1, interleukin-6 (IL-6), and IL-8. FSL-1 also activated macrophages to produce tumor necrosis factor alpha as the Mycoplasma fermentansderived lipopeptide MALP-2 (Pam 2 CGNNDESNISFKEK), a potent macrophage-activating lipopeptide, did. The level of the activity of FSL-1 was higher than that of MALP-2. This result suggests that the difference in the amino acid sequence of the peptide portion affects the activity because the framework structure other than the amino acid sequence of the former is the same as that of the latter. To determine minimal structural requirements for the activity of FSL-1, the diacylglyceryl Cys and the peptide portions were examined for this activity. Both portions did not reveal the activity. A single amino acid substitution from Phe to Arg and a fatty acid substitution from palmitic acid to stearic acid drastically reduced the activity. Similar results were obtained in measuring the NF-B reporter activity of FSL-1 to human embryonic kidney 293 cells transfected with Toll-like receptor 2 and 6, together with a NF-B-dependent luciferase reporter plasmid. These results suggest that both the diacylglyceryl and the peptide portions of FSL-1 are indispensable for the expression of biological activities and for the recognition by Toll-like receptors 2 and 6 and that the recognition of FSL-1 by Toll-like receptors 2 and 6 appears to be hydrophobic.Various bacterial cell wall components such as lipopolysaccharides (LPS), lipoteichoic acid, peptidoglycans, and lipoproteins (LP) have been shown to activate macrophages, fibroblasts, or lymphocytes to induce production of cytokines (16). Escherichia coli LP were first characterized and sequenced by Braun (9), and they have been demonstrated to be biologically active (5)(6)(7)(8)20). The part of LP responsible for biological activity is the N-terminal lipopeptide moiety, the structure of which is S-(2,3-bispalmitoyloxypropyl)-N-palmitoyl-Cys-SerSer-Asp-Ala-(Pam 3 CSNNA-) (7).Mycoplasmas, wall-less microorganisms, also possess LP capable of activating macrophages or fibroblasts (11,27,28,31,32). Mühlradt et al. (27,28) recently identified a 2-kDa lipopeptide, MALP-2, from Mycoplasma fermentans that is capable of activating monocytes/macrophages, and these authors determined the structure to be S-(2,3-bispalmitoyloxypropyl) Cys-Gly-Asn-Asn-Asp-Glu-Ser-Asn-Ile-Ser-Phe-Lys-Glu-Lys (Pam 2 CGNNDESNISFKEK). We have also found that Mycoplasma salivarium LP activate normal human gingival fibroblasts (HGF) to induce production of inflammatory cytokines and surface expression of ICAM-1 and have purified a 44-kDa LP (LP44) responsible for the activity (32). The structure of the N-ter...

Background and PurposeThe Japanese Clinical Practice Guidelines for Management of Sepsis and Septic Shock 2016 (J‐SSCG 2016), a Japanese‐specific set of clinical practice guidelines for sepsis and septic shock created jointly by the Japanese Society of Intensive Care Medicine and the Japanese Association for Acute Medicine, was first released in February 2017 in Japanese. An English‐language version of these guidelines was created based on the contents of the original Japanese‐language version.MethodsMembers of the Japanese Society of Intensive Care Medicine and the Japanese Association for Acute Medicine were selected and organized into 19 committee members and 52 working group members. The guidelines were prepared in accordance with the Medical Information Network Distribution Service (Minds) creation procedures. The Academic Guidelines Promotion Team was organized to oversee and provide academic support to the respective activities allocated to each Guideline Creation Team. To improve quality assurance and workflow transparency, a mutual peer review system was established, and discussions within each team were open to the public. Public comments were collected once after the initial formulation of a clinical question (CQ), and twice during the review of the final draft. Recommendations were determined to have been adopted after obtaining support from a two‐thirds (>66.6%) majority vote of each of the 19 committee members.ResultsA total of 87 CQs were selected among 19 clinical areas, including pediatric topics and several other important areas not covered in the first edition of the Japanese guidelines (J‐SSCG 2012). The approval rate obtained through committee voting, in addition to ratings of the strengths of the recommendation and its supporting evidence were also added to each recommendation statement. We conducted meta‐analyses for 29 CQs. Thirty seven CQs contained recommendations in the form of an expert consensus due to insufficient evidence. No recommendations were provided for 5 CQs.ConclusionsBased on the evidence gathered, we were able to formulate Japanese‐specific clinical practice guidelines that are tailored to the Japanese context in a highly transparent manner. These guidelines can easily be used not only by specialists, but also by non‐specialists, general clinicians, nurses, pharmacists, clinical engineers, and other healthcare professionals.

BackgroundThe options for medical use of signaling molecules as stimulators of tissue regeneration are currently limited. Preclinical evidence suggests that fibroblast growth factor (FGF)-2 can promote periodontal regeneration. This study aimed to clarify the activity of FGF-2 in stimulating regeneration of periodontal tissue lost by periodontitis and to evaluate the safety of such stimulation.Methodology/Principal FindingsWe used recombinant human FGF-2 with 3% hydroxypropylcellulose (HPC) as vehicle and conducted a randomized double-blinded controlled trial involving 13 facilities. Subjects comprised 74 patients displaying a 2- or 3-walled vertical bone defect as measured ≥3 mm apical to the bone crest. Patients were randomly assigned to 4 groups: Group P, given HPC with no FGF-2; Group L, given HPC containing 0.03% FGF-2; Group M, given HPC containing 0.1% FGF-2; and Group H, given HPC containing 0.3% FGF-2. Each patient underwent flap operation during which we administered 200 µL of the appropriate investigational drug to the bone defect. Before and for 36 weeks following administration, patients underwent periodontal tissue inspections and standardized radiography of the region under investigation. As a result, a significant difference (p = 0.021) in rate of increase in alveolar bone height was identified between Group P (23.92%) and Group H (58.62%) at 36 weeks. The linear increase in alveolar bone height at 36 weeks in Group P and H was 0.95 mm and 1.85 mm, respectively (p = 0.132). No serious adverse events attributable to the investigational drug were identified.ConclusionsAlthough no statistically significant differences were noted for gains in clinical attachment level and alveolar bone gain for FGF-2 groups versus Group P, the significant difference in rate of increase in alveolar bone height (p = 0.021) between Groups P and H at 36 weeks suggests that some efficacy could be expected from FGF-2 in stimulating regeneration of periodontal tissue in patients with periodontitis.Trial RegistrationClinicalTrials.gov NCT00514657

Early stationary phase culture supernatants of Streptomyces coelicolor A3(2) contained at least four small diffusible signaling molecules that could elicit precocious antibiotic synthesis in the producing strain. The compounds were not detected in exponentially growing cultures. One of these compounds, SCB1, was purified to homogeneity and shown to be a ␥-butyrolactone of structure (2R,3R,1R)-2-(1-hydroxy-6-methylheptyl)-3-hydroxymethylbutanolide. Bioassays of chemically synthesized SCB1, and of its purified stereoisomers, suggest that SCB1 acts in a highly specific manner to elicit the production of both actinorhodin and undecylprodigiosin, the two pigmented antibiotics made by S. coelicolor.Small diffusible signaling molecules play regulatory roles in a wide variety of bacteria. The most intensively studied are the N-acyl homoserine lactones, which have diverse roles as signaling molecules in a wide range of Gram-negative bacteria (1). Among Gram-positive bacteria, structurally similar but chemically distinct ␥-butyrolactones play determining roles in antibiotic production and sporulation in Streptomyces species (2) (Fig. 1). Streptomycetes are mycelial soil bacteria with a developmental program that results in sporulation. They also produce a wide variety of antibiotics with important uses in medicine and in agriculture. These antibiotics are the products of complex biosynthetic pathways, activated typically in a growth phase-dependent manner (3). In liquid culture, antibiotic production generally occurs in stationary phase (4, 5), while in surface-grown cultures, it usually coincides with the onset of morphological differentiation, i.e. the formation of aerial hyphae.The most intensively studied ␥-butyrolactone is A-factor (2-isocapryloyl-3R-hydroxymethyl-␥-butyrolactone), which is required for streptomycin production and sporulation in Streptomyces griseus (6). A-factor binds to a cytoplasmic protein that in its absence represses streptomycin production and morphological differentiation (7). Other ␥-butyrolactones have also been shown to induce antibiotic biosynthesis, e.g. the virginiae butanolides of Streptomyces virginiae (8, 9). Moreover, since at least 60% of Streptomyces species appear to produce ␥-butyrolactones (9), these compounds are likely to play important roles as extracellular signaling molecules in the biology of these organisms. Streptomyces coelicolor A3(2), the most genetically characterized streptomycete, produces at least four antibiotics, including the blue-pigmented polyketide actinorhodin (Act), 1 and the red-pigmented tri-pyrolle undecylprodigiosin (Red), both of which are produced in a growth phase-dependent manner (4, 5, 10). Earlier studies (11, 12) led to the isolation and partial structural determination of six ␥-butyrolactones made by S. coelicolor (Fig. 2), but there was no report of their biological activity in the producing strain.This paper identifies four low molecular weight compounds present in the supernatants of transition and stationary phase cultures of S. coelicolor...

Toll-like receptors (TLRs) 2 and 4 have recently been identified as possible signal transducers for various bacterial ligands. To investigate the roles of TLRs in the recognition of periodontopathic bacteria by the innate immune system, a Chinese hamster ovary (CHO) nuclear factor-B (NF-B)-dependent reporter cell line, 7.7, which is defective in both TLR2-and TLR4-dependent signaling pathways was transfected with human CD14 and TLRs. When the transfectants were exposed to freeze-dried periodontopathic bacteria, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Capnocytophaga ochracea, and Fusobacterium nucleatum, and a non-oral bacterium, Escherichia coli, all species of the bacteria induced NF-B-dependent CD25 expression in 7.7/huTLR2 cells. Although freeze-dried A. actinomycetemcomitans, F. nucleatum, and E. coli also induced CD25 expression in 7.7/huTLR4 cells, freeze-dried P. gingivalis did not. Similarly, lipopolysaccharides (LPS) extracted from A. actinomycetemcomitans, F. nucleatum, and E. coli induced CD25 expression in 7.7/huTLR4 cells, but LPS from P. gingivalis and C. ochracea did not. Furthermore, LPS from P. gingivalis and C. ochracea attenuated CD25 expression in 7.7/huTLR4 cells induced by repurified LPS from E. coli. LPS from P. gingivalis and C. ochracea also inhibited the secretion of interleukin-6 (IL-6) from U373 cells, the secretion of IL-1␤ from human peripheral blood mononuclear cells, and ICAM-1 expression in human gingival fibroblasts induced by repurified LPS from E. coli. These findings indicated that LPS from P. gingivalis and C. ochracea worked as antagonists for human TLR4. The antagonistic activity of LPS from these periodontopathic bacteria may be associated with the etiology of periodontal diseases.