Cyclic compressive loading on 3D tissue of human synovial fibroblasts upregulates prostaglandin E2 via COX-2 production without IL-1ß and TNF-α.

OBJECTIVE: Excessive mechanical stress on synovial joints causes osteoarthritis (OA) and results in the production of prostaglandin E2 (PGE2), a key molecule in arthritis, by synovial fibroblasts. However, the relationship between arthritis-related molecules and mechanical stress is still unclear. The purpose of this study was to examine the synovial fibroblast response to cyclic mechanical stress using an in vitro osteoarthritis model. METHOD: Human synovial fibroblasts were cultured on collagen scaffolds to produce three-dimensional constructs. A cyclic compressive loading of 40 kPa at 0.5 Hz was applied to the constructs, with or without the administration of a cyclooxygenase-2 (COX-2) selective inhibitor or dexamethasone, and then the concentrations of PGE2, interleukin-1ß (IL-1ß), tumour necrosis factor-α (TNF-α), IL-6, IL-8 and COX-2 were measured. RESULTS: The concentrations of PGE2, IL-6 and IL-8 in the loaded samples were significantly higher than those of unloaded samples; however, the concentrations of IL-1ß and TNF-α were the same as the unloaded samples. After the administration of a COX-2 selective inhibitor, the increased concentration of PGE2 by cyclic compressive loading was impeded, but the concentrations of IL-6 and IL-8 remained high. With dexamethasone, upregulation of PGE2, IL-6 and IL-8 was suppressed. CONCLUSION: These results could be useful in revealing the molecular mechanism of mechanical stress in vivo for a better understanding of the pathology and therapy of OA. Cite this article: Bone Joint Res 2014;3:280-8.

The use of MRI in pre-operative evaluation of anterior talofibular ligament in chronic ankle instability.

OBJECTIVES: To evaluate the applicability of MRI for the quantitative assessment of anterior talofibular ligaments (ATFLs) in symptomatic chronic ankle instability (CAI). METHODS: Between 1997 and 2010, 39 patients with symptomatic CAI underwent surgical treatment (22 male, 17 female, mean age 25.4 years (15 to 40)). In all patients, the maximum diameters of the ATFLs were measured on pre-operative T2-weighted MR images in planes parallel to the path of the ATFL. They were classified into three groups based on a previously published method with modifications: 'normal', diameter = 1.0 - 3.2 mm; 'thickened', diameter > 3.2 mm; 'thin or absent', diameter < 1.0 mm. Stress radiography was performed with the maximum manual force in inversion under general anaesthesia immediately prior to surgery. In surgery, ATFLs were macroscopically divided into two categories: 'thickened', an obvious thickened ligament and 'thin or absent'. The imaging results were compared with the macroscopic results that are considered to be of a gold standard. RESULTS: Agreement was reached when comparison was made between groups, based on MRI and macroscopic findings. ATFLs were abnormal in all 39 cases and classified as ten 'thickened' and 29 'thin or absent'. As to talar tilt stress radiography, a clear cut-off angle, which would allow discrimination between 'thickened' and 'thin or absent' patients, was not identified. CONCLUSION: MRI is valuable as a pre-operative assessment tool that can provide the quantitative information of ATFLs in patients with CAI. Cite this article Bone Joint Res 2014;3:241-5.

Virtual CT colectomy by three-dimensional imaging using multidetector-row CT for laparoscopic colorectal surgery.

Laparoscopic colorectal surgery has been attracting attention for its capacity to improve the quality of life (QOL) of patients. However, there are disadvantages to this approach, namely, it is difficult to obtain an image of the entire view of the operative field, and organs and lesions cannot be manipulated directly by the surgeon during surgery. For this reason, it takes a relatively large amount of time to ligate vessel, which can vary between patients. Furthermore, vessels and organs can be damaged during lymph nodes dissection under laparoscopic guidance, leading to heavy bleeding that prevents the surgeon from having access to a good view of the operative field. Then, to assess preoperatively the vascular anatomy, we carried out multiphase, contrast-enhanced examinations using multidetector-row CT (MDCT) on patients with colorectal cancer, and prepared the fused image of 3D images of arteries, veins, the colorectum, organs, and tumor. We called the utilization of 3D imaging virtual CT colectomy, which contributed to rapid and safe manipulation of the origins of the arteries and the veins, as well as lymph nodes dissection, without incurring injury to the involved arteries and veins.

Synthesis and evaluation of bifunctional anti-HIV agents based on specific CXCR4 antagonists-AZT conjugation.

We have previously found that T140, a 14-amino acid residue peptide, inhibits infection of target cells by T cell-line-tropic strains of HIV-1 (X4-HIV-1) through its specific binding to a chemokine receptor, CXCR4. Here, we report synthesis and evaluation of bifunctional anti-HIV compounds, which are composed of T140 analogues and a reverse transcriptase inhibitor, 3'-azido-3'-deoxythymidine (AZT). Novel conjugated analogues have been proved to have the ability for controlled release of AZT in neutral aqueous media as well as mouse and feline sera, and high selectivity indexes (SIs, 50% cytotoxic concentration/50% effective concentration) caused by a synergistic effect of two different regenerating agents. Thus, these bifunctional compounds have several potential advantages. T140 analogues can possibly work as a carrier of AZT targeting T cells due to their specific affinity for CXCR4 on T cells. A synergistic effect by two types of regenerating agents may enable drug dosage to be reduced, and thus it may effectively suppress toxic side effects and the appearance of drug-resistant virus.

Development of specific CXCR4 inhibitors possessing high selectivity indexes as well as complete stability in serum based on an anti-HIV peptide T140.

We previously reported a truncated polyphemusin peptide analogue, T140, which efficiently inhibits infection of target cells by T-cell line-tropic strains of HIV-1 (X4-HIV-1) through its specific binding to a chemokine receptor, CXCR4. We have found that T140 is not stable in feline serum due to the cleavage of the C-terminal Arg,(14) indispensable for anti-HIV activity. On the other hand, a C-terminally amidated analogue of T140, TZ14004, has been found to be completely stable in incubation in the serum for 2 days. The C-terminal amide is thought to be needed for stability in serum. However, TZ14004 does not have fairly strong anti-HIV activity, but has relatively strong cytotoxicity, probably due to an increase by +1 charge from total +7 charges of T140. In our previous study, the number of total +6 charges seemed to be a suitable balance between activity and cytotoxicity. In this study, we have conducted a double-L-citrulline (Cit)-scanning study on TZ14004 based on the C-terminally amidated form in due consideration of the total net charges in the whole molecule to find novel effective CXCR4 inhibitors, TN14003 ([Cit(6)]-T140 with the C-terminal amide) and TC14012 ([Cit(6), D-Cit(8)]-T140 with the C-terminal amide), which possess high selectivity indexes (SIs) and complete stability in feline serum.

Increase of R5 HIV-1 infection and CCR5 expression in T cells treated with high concentrations of CXCR4 antagonists and SDF-1.

The chemokine receptors CXCR4 and CCR5 are considered to be potential targets for the inhibition of HIV-1 replication. We found that the synthetic peptides T134 and T140 (see text for full names) inhibited X4 HIV-1 infection with selectivity and low toxicity because they acted as CXCR4 antagonists. However, high concentrations of T134, T140, and ALX40-4C (see text for full name) increased the expression of CCR5 and R5 HIV-1 infection, as did stromal cell-derived factor 1 (SDF-1). In contrast to CXCR4 antagonists and SDF-1, viral monocyte inflammatory protein (vMIP) II inhibited not only anti-CXCR4 monoclonal antibody (MAb) but also inhibited anti-CCR5 MAb binding to human peripheral blood mononuclear cells, and inhibited both X4 and R5 HIV-1 strains. T134, T140, ALX40-4C, and SDF-1 increased viral transcription in the treated cells. In addition, ALX40-4C and SDF-1 also increased nuclear transcription factor (NF)-kappaB. However, the mechanisms of action of T134 and T140 are different from those of clinically used anti-HIV drugs. Thus, synergistic activities were observed in the concomitant treatment with T134 and reverse transcriptase inhibitors or protease inhibitors. Our findings, presented here, are noteworthy in regard to the potential clinical use of these agents as drugs for the treatment of AIDS.

Diverse biological activity of polycaphenol.

Millimolar concentrations of alkaline extract of Cacao husk (polycaphenol) were more cytotoxic to human oral tumor cells (human oral squamous cell carcinoma HSC-2, human salivary gland tumor HSG), than to human gingival fibroblast (HGF), suggesting its tumor-specific action. Polycaphenol enhanced the radical intensity and cytotoxic activity of vitamin K3 more effectively than that of sodium ascorbate (vitamin C). Polycaphenol effectively scavenged the superoxide anion, produced by the hypoxanthine-xanthine oxidase reaction, indicating bimodal (prooxidant and antioxidant) action of polycaphenol. Polycaphenol inhibited the cytopathic effect of HIV (human immunodeficiency virus) infection in MT-4 cells, to a comparable extent as that achieved by lignin. Pretreatment of mice with polycaphenol protected them from lethal infection of Eschericia coli. These data suggest the medicinal efficacy of polycaphenol.

Anti-human immunodeficiency virus activity of YK-FH312 (a betulinic acid derivative), a novel compound blocking viral maturation.

Betulinic acid, a triterpenoid isolated from the methyl alcohol extract of the leaves of Syzigium claviflorum, was found to have a potent inhibitory activity against human immunodeficiency virus type 1 (HIV-1). Betulinic acid derivatives were synthesized to enhance the anti-HIV activity. Among the derivatives, 3-O-(3',3'-dimethylsuccinyl) betulinic acid, designated YK-FH312, showed the highest activity against HIV-induced cytopathic effects in HIV-1-infected MT-4 cells. To determine the step(s) of HIV replication affected by YK-FH312, a syncytium formation inhibition assay in MOLT-4/HIV-1(IIIB) and MOLT-4 coculture, a multinuclear-activation-of-galactosidase-indicator (MAGI) assay in MAGI-CCR5 cells, electron microscopic observation, and a time-of-addition assay were performed. In the syncytium formation inhibition assay or in the MAGI assay for de novo infection, the compound did not show inhibitory effects against HIV replication. Conversely, no virions were detected in HIV-1-infected cell cultures treated with YK-FH312 either by electron microscopic observation or by viral yield in the supernatant. In accordance with a p24 enzyme-linked immunosorbent assay of culture supernatant in the time-of-addition assay, YK-FH312 inhibited virus expression in the supernatant when it was added 18 h postinfection. However, Western blot analysis of the cells in the time-of-addition assay revealed that the production of viral proteins in the cells was not inhibited completely by YK-FH312. These results suggest that YK-FH312 might affect the step(s) of virion assembly and/or budding of virions, and this is a novel mechanism of action of an anti-HIV compound.

Antioxidative activity of Allium victorialis L. extracts.

Allium victorialis L. (Liliaceae, "Hon-Gyoujya Nin-Niku" in Japanese) was successively extracted with hexane, acetone, methanol and 70% methanol and the extracts were further separated into a total of twenty-five fractions by silica gel and ODS column chromatographies. The biological activities of these four extracts and 25 column fractions were compared. The cytotoxic activity of all extracts and fractions against two oral tumor cell lines was significantly higher than that against normal human gingival fibroblasts, suggesting their tumor-specific action. Three methanol column fractions [M2, M3, M6] and a 70% methanol column fraction [70M6] most effectively reversed the multidrug resistance (MDR) against L5178 mouse T cell lymphoma. The electron spin resonance (ESR) spectroscopy showed that methanol column fractions and 70% methanol extracts produced the highest amount of radical(s) and most efficiently scavenging O2*-, generated by the hypoxanthine-xanthine reaction system, suggesting that the same substances in these fractions display both prooxidant and antioxidant properties. They showed no anti-human immunodeficiency virus (HIV) or anti-Helicobacterpylori activity. These data suggest the medicinal efficacy of Allium victorialis extract.

Pharmacophore identification of a specific CXCR4 inhibitor, T140, leads to development of effective anti-HIV agents with very high selectivity indexes.

A polyphemusin peptide analogue, T22 ([Tyr(5,12), Lys7]-polyphemusin II), and its shortened potent analogues, T134 (des-[Cys(8,13), Tyr(9,12)]-[D-Lys10, Pro11, L-citrulline16]-T22 without C-terminal amide) and T140 [[L-3-(2-naphthyl)alanine3]-T134], strongly inhibit the T-cell line-tropic (T-tropic) HIV-1 infection through their specific binding to a chemokine receptor, CXCR4. T22 is an extremely basic peptide possessing five Arg and three Lys residues in the molecule. In our previous study, we found that there is an apparent correlation in the T22-related peptides between the number of total positive charges and anti-HIV activity or cytotoxicity. Here, we have conducted the conventional Ala-scanning study in order to define the anti-HIV activity pharmacophore of T140 (the strongest analogue among our compounds) and identified four indispensable amino acid residues (Arg2, Nal3, Tyr5, and Arg14). Based on this result, a series of L-citrulline (Cit)-substituted analogues of T140 with decreased net positive charges have been synthesized and evaluated in terms of anti-HIV activity and cytotoxicity. As a result, novel effective inhibitors, TC14003 and TC14005, possessing higher selectivity indexes (SIs, 50% cytotoxic concentration/50% effective concentration) than that of T140 have been developed.

Role of apoptosis signal-regulating kinase in regulation of the c-Jun N-terminal kinase pathway and apoptosis in sympathetic neurons.

We have previously shown that nerve growth factor (NGF) withdrawal-induced death requires the activity of the small GTP-binding protein Cdc42 and that overexpression of an active form of Cdc42 is sufficient to mediate neuronal apoptosis via activation of the c-Jun pathway. Recently, a new mitogen-activated protein (MAP) kinase kinase kinase, apoptosis signal-regulating kinase 1 (ASK1) which activates both the c-Jun N-terminal kinase (JNK) and p38 MAP kinase pathways and plays pivotal roles in tumor necrosis factor- and Fas-induced apoptosis, has been identified. Therefore, we investigated the role of ASK1 in neuronal apoptosis by using rat pheochromocytoma (PC12) neuronal cells and primary rat sympathetic neurons (SCGs). Overexpression of ASK1-DeltaN, a constitutively active mutant of ASK1, activated JNK and induced apoptosis in differentiated PC12 cells and SCG neurons. Moreover, in differentiated PC12 cells, NGF withdrawal induced a four- to fivefold increase in the activity of endogenous ASK1. Finally, expression of a kinase-inactive ASK1 significantly blocked both NGF withdrawal- and Cdc42-induced death and activation of c-jun. Taken together, these results demonstrate that ASK1 is a crucial element of NGF withdrawal-induced activation of the Cdc42-c-Jun pathway and neuronal apoptosis.

Pathogenicity of Corynebacterium kutscheri in the Syrian hamster.

The pathogenicity of Corynebacterium kutscheri isolated for the first time from Syrian hamster was experimentally studied in hamsters. In hamsters given intramuscular (i.m.) or subcutaneous (s.c.) inoculation with 10 or 10(3) bacteria, neither clinical signs nor gross lesions were found. In those given 10(5) bacteria i.m., moderate proliferation of granulation tissue was found in the muscle of the inoculation region at necropsy. In the animals given 10(5) bacteria s.c., a nodular lesion was observed at the inoculation site 2 days post-inoculation (p.i.), but the nodules subsided gradually from 6 days p.i. and were unclear 10 days p.i. At necropsy, small abscesses were found in all the animals in this group. In those given 10(7) bacteria either i.m. or s.c., lesions were clearly observed at the inoculation site 1 to 10 days p.i., and a large abscess was noted at necropsy. The organisms were isolated only from the lesions in the groups. Agglutinating antibody in the sera was detected only in the animals given 10(5) or 10(7) bacteria. This suggests that 10(5) of C. kutscheri are needed to form localized nodular abscesses in Syrian hamsters.

Immunogenicity and protective effect against oral colonization by Streptococcus mutans of synthetic peptides of a streptococcal surface protein antigen.

Streptococcus mutans is known to be a major causative organism of human dental caries. A surface protein Ag with a molecular mass of 190 kDa of S. mutans (PAc) is receiving attention as an anticaries vaccine. We have recently determined the complete nucleotide sequence of the gene for PAc. In this study, four peptides were synthesized on the basis of amino acid sequence of PAc. Among these peptides, PAc(301-319) corresponding to the alanine-rich repeating amino acid region was the most strongly bound by polyclonal murine anti-rPAc antibodies. The peptide partially inhibited the binding of polyclonal anti-rPAc antibodies to rPAc. The peptide induced the proliferation of T cells from BALB/c mice immunized with rPAc. Subcutaneous immunization with PAc(301-319) or rPAc emulsified in CFA/IFA induced high serum IgG responses to rPAc and PAc(301-319). In addition, serum IgG responses to a surface protein Ag with a molecular mass of 210 kDa of Streptococcus sobrinus were elicited in mice immunized by s.c. injection with PAc(301-319) or rPAc. Intranasal immunization with PAc(301-319) coupled to cholera toxin B subunit (CTB) or with rPAc and free CTB induced high serum IgG responses to rPAc. The immunization with PAc(301-319) coupled to CTB or rPAc and free CTB suppressed the colonization of murine teeth by S. mutans. These results suggest that intranasal immunization with the peptide or rPAc may be effective for the prevention of dental caries.

Oral immunization with recombinant Streptococcus lactis carrying the Streptococcus mutans surface protein antigen gene.

A recombinant Streptococcus lactis strain which carries the structural gene for a surface protein antigen (PAc) of 190,000 daltons from Streptococcus mutans serotype c was constructed for development of an oral vaccine against dental caries. The gene from S. mutans MT8148 joined to shuttle vector pSA3 was successfully transformed into S. lactis IL1403. A small amount of PAc was detected in the cell homogenate and cytoplasmic fraction of the recombinant S. lactis, but not in the culture supernatant of the recombinant, by Western immunoblotting and dot immunoblotting. The level of PAc-specific mRNA in the recombinant strain was lower than that in S. mutans MT8148. However, significant salivary immunoglobulin A and serum immunoglobulin G responses to PAc were induced in mice immunized orally with the recombinant S. lactis.

Surface hydrophobicity, adherence, and aggregation of cell surface protein antigen mutants of Streptococcus mutans serotype c.

The pac gene of the serotype c strain Streptococcus mutans MT8148 encodes a cell surface protein antigen (PAc) of approximate 190 kilodaltons. The serotype c strain S. mutans GS-5 does not produce the 190-kilodalton PAc but produces a lower-molecular-weight protein that reacts with anti-PAc serum. The SphI-BamHI fragment of the pac gene was ligated with the S. mutans-Escherichia coli shuttle vector pSA3. The chimeric shuttle vector was transformed into strain GS-5, and two transformants (TK15 and TK18) were isolated. These transformants produced a large amount of cell-free and cell-bound PAc of 190 kilodaltons. No plasmid was isolated from these transformants, and the EcoRI fragments of their chromosomal DNA hybridized with the erythromycin resistance gene in the shuttle vector DNA, indicating insertion of the chimeric shuttle vector DNA into the chromosomal DNA. The cell hydrophobicity of strains TK15 and TK18 as well as PAc-defective mutants constructed by inserting an erythromycin resistance gene into the pac gene of strain MT8148 was analyzed. Strains MT8148, TK15, and TK18 were hydrophobic. On the other hand, strain GS-5 and PAc-defective MT8148 transformants were hydrophilic. Resting cells of the hydrophobic strains attached in larger numbers to saliva-coated hydroxyapatite than did the hydrophilic strains. Human whole saliva induced the aggregation of cells of the hydrophobic strains but not that of cells of the hydrophilic strains. These results suggest that cell surface PAc of S. mutans serotype c participates in attachment of the streptococcal cell to experimental pellicles.