Immune-bone interplay in the structural damage in rheumatoid arthritis.

The immune and bone systems maintain homeostasis by interacting closely with each other. Rheumatoid arthritis is a pathological consequence of their interplay, as activated T cell immune responses result in osteoclast-mediated bone erosion. An imbalance between forkhead box protein 3 (Foxp3)+ regulatory T (Treg ) cells and T helper type 17 (Th17) cells is often linked with autoimmune diseases, including arthritis. Th17 cells contribute to the bone destruction in arthritis by up-regulating receptor activator of nuclear factor kappa-Β ligand (RANKL) on synovial fibroblasts as well as inducing local inflammation. Studies on the origin of Th17 cells in inflammation have shed light on the pathogenic conversion of Foxp3+ T cells. Th17 cells converted from Foxp3+ T cells (exFoxp3 Th17 cells) comprise the most potent osteoclastogenic T cell subset in inflammatory bone loss. It has been suggested that osteoclastogenic T cells may have developed originally to stop local infection in periodontitis by inducing tooth loss. In addition, Th17 cells also contribute to the pathogenesis of arthritis by modulating antibody function. Antibodies and immune complexes have attracted considerable attention for their direct role in osteoclastogenesis, and a specific T cell subset in joints was shown to be involved in B cell antibody production. Here we summarize the recent advances in our understanding of the immune-bone interplay in the context of the bone destruction in arthritis.

MSC/ECM Cellular Complexes Induce Periodontal Tissue Regeneration.

Transplantation of mesenchymal stem cells (MSCs), which possess self-renewing properties and multipotency, into a periodontal defect is thought to be a useful option for periodontal tissue regeneration. However, developing more reliable and predictable implantation techniques is still needed. Recently, we generated clumps of an MSC/extracellular matrix (ECM) complex (C-MSC), which consisted of cells and self-produced ECM. C-MSCs can regulate their cellular functions in vitro and can be grafted into a defect site, without any artificial scaffold, to induce bone regeneration. Accordingly, this study aimed to evaluate the effect of C-MSC transplantation on periodontal tissue regeneration in beagle dogs. Seven beagle dogs were employed to generate a premolar class III furcation defect model. MSCs isolated from dog ilium were seeded at a density of 7.0 × 104 cells/well into 24-well plates and cultured in growth medium supplemented with 50 µg/mL ascorbic acid for 4 d. To obtain C-MSCs, confluent cells were scratched using a micropipette tip and were then torn off as a cellular sheet. The sheet was rolled up to make round clumps of cells. C-MSCs were maintained in growth medium or osteoinductive medium (OIM) for 5 or 10 d. The biological properties of C-MSCs were evaluated in vitro, and their periodontal tissue regenerative activity was tested by using a dog class III furcation defect model. Immunofluorescence analysis revealed that type I collagen fabricated the form of C-MSCs. OIM markedly elevated calcium deposition in C-MSCs at day 10, suggesting its osteogenic differentiation capacity. Both C-MSCs and C-MSCs cultured with OIM transplantation without an artificial scaffold into the dog furcation defect induced periodontal tissue regeneration successfully compared with no graft, whereas osteogenic-differentiated C-MSCs led to rapid alveolar bone regeneration. These findings suggested that the use of C-MSCs refined by self-produced ECM may represent a novel predictable periodontal tissue regenerative therapy.

Radical modulation activity of pine cone extracts of Pinus elliottii var. Elliottii.

The radical modulation activity of lignins prepared from the cone of Pinus elliottii var. Elliottii was investigated, using ESR spectroscopy. These lignins produced radical(s) under alkaline conditions, and the radical intensity was increased with increasing pH. Lower concentrations of lignins slightly reduced the radical intensity of sodium ascorbate, whereas higher concentrations of lignins enhanced both the radical intensity and cytotoxic activity of sodium ascorbate. Lignins effectively scavenged superoxide anion, produced by hypoxanthine-xanthine oxidase reaction. Elliottii lignins significantly inhibited the human immunodeficiency virus (HIV)-induced cytopathic effect, in similar fashions to other natural, commercial and synthetic lignins. Pretreatment of mice with lignins significantly protected them from the lethal infection with E. coli. Crude alkaline extracts of Elliottii pine cone displayed similar magnitude of activity with lignins. These data further supports the medicinal efficacy of plant extracts.

Antitumor, antiviral and immunopotentiating activities of pine cone extracts: potential medicinal efficacy of natural and synthetic lignin-related materials (review).

Several antitumor substances that effectively inhibited the growth of ascites and solid tumor cells transplanted in mice were isolated from pine cone NaOH extract by acid- and ethanol-precipitation. These antitumor substances were also potent antiviral agents against human immunodeficiency virus, herpes simplex virus and influenza virus; they induced antimicrobial activity against Staphylococcal aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Candida albicans, and induced antiparasite activity against Hymenolepis nana in mice. Chemical analysis of these substances by IR, UV, NMR, ESR and partition chromatography on cellulose-TLC plate disclosed that they had lignin-related structures complexed with sugars or polysaccharides. Chlorinated decomposition of the lignin portion significantly reduced their antiviral activity. In agreement with this, the antiviral activity of synthesized lignins prepared by polymerization of phenylpropanoid precursors was comparable to that of the undecomposed counterparts of the pine cone extract. Acid hydrolysis of the polysaccharide portion significantly reduced the ability of the substances to induce antitumor and antimicrobial activities in mice. With an appropriate eliciting agent, intravenous administration of natural lignified substances transiently induced endogenous production of a cytotoxic factor (possibly tumor necrosis factor) in normal mice. Their priming activity was significantly higher than that of their component units or degradation products. These data suggest the importance of conjugating lignins with polysaccharides for in vivo expression of various kinds of immunopotentiating activity. As possible explanations for their induction of a variety of immunopotentiating activities, these natural and synthetic lignins stimulated macrophage NBT-reducing activity, polymorphonuclear cell (PMN) iodination and splenocyte DNA synthesis and inhibited poly (ADP-ribose) glycohydrolase, RNA-dependent DNA polymerase (reverse transcriptase) and RNA-dependent RNA polymerase activities.

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.

Antimicrobial spectrum of lignin-related pine cone extracts of Pinus parviflora Sieb. et Zucc.

Pine cones of Pinus parviflora Sieb., et Zucc, were extracted successively with 5% NaHCO3, 3% NH4OH, 1% NaOH and 4% NaOH, and the extracts were tested for ability to induce antimicrobial activity in mice infected with Staphylococcal aureus. Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Candida albicans, or Salmonella enteritidis. These alkaline extracts were potent against the first 5 of these strains of microorganisms, and the activity was recovered mostly from their acid-precipitates at pH 5. After further fractionation on a Sephadex LH-60 column, the highest molecular weight fraction was most potent. Chemical structures in these bioactive fractions were assumed to be lignin-related structures, based on spectral data from UV, NMR and IR spectroscopy.

Lignified materials as potential medicinal resources. II. Prevention of pathogenic bacterial infections in mice.

Antibacterial activity was demonstrated when ddY mice were intraperitoneally infected with E. coli or P. aeruginosa two days after a single ip-injection of a hot-water or alkaline extract obtained from lignified materials, such as chips of slash pine, Douglas fir, tallow wood, and two shelf fungi. Lignified material is a promising medicinal resource for the prevention and/or treatment of pathogenic microbial infection.

Diverse biological activities of moxa extract and smoke.

Diverse biological activities of moxa extracts and smoke (gas phase) were investigated. Moxa was extracted with hot water (Fr. I), or ethanol (Fr. II), or extracted with hot water after ethanol wash (Fr. III) and then lyophilized to obtain the dried powders. Moxa smoke (containing a lot of gaseous components obtained by burning Moxa) (Fr. IV) was collected into phosphate-buffered saline and quantified spectrophotometrically. These extracts and Moxa smoke showed comparable cytotoxic activity against human oral tumor cell lines (HSC-2, HSG). Human gingival fibroblasts (HGF) were more resistant to any Moxa fractions. Neither of the extracts showed anti-HIV activity. Pretreatment of mice with Fr. I significantly reduced the lethal effect of E. coli infection. All extracts produced radicals under alkaline condition, with a maximum intensity at pH 10.5, and enhanced the radical intensity of sodium ascorbate. It was unexpected that these extracts show significant O2- scavenging activities. These data suggest the medicinal efficacy of Moxa extracts and smoke.

Radical modulation activity of lignins from a mangrove plant, Ceriops decandra (Griff.) Ding Hou.

The radical modulation activity of hot water and alkaline extracts from leaf of Ceriops decandra, a mangrove plant, was investigated using ESR spectroscopy. IR and NMR analyses demonstrate that the leaf extracts have a lignin-like polyphenolic structure. All these extracts produced radical(s) under alkaline conditions. The radical intensity of sodium ascorbate was slightly reduced at lower concentrations of the extracts, but it was synergistically enhanced at higher concentrations. All the extracts effectively scavenged superoxide anion, produced by hypoxanthine-xanthine oxidase reaction. Pretreatment of mice with the extracts significantly protected them from the lethal infection by E. coli. Similar activity was found in lignins from pine seed shell of Pinus parviflora Sieb. et Zucc. These data further support the medicinal efficacy of plant extracts.

Double replica electroblotting for the simultaneous phenotyping of C6 and C7, and its application to the examination of bloodstains.

A new reliable and reproducible technique for the simultaneous determination of C6 and C7 types is presented, which employs double replica electroblotting after isoelectric focusing. It permitted clear discrimination of both C6 and C7 components, and the patterns were nearly comparable to those demonstrated separately. The population data obtained by this new technique fitted the genetic hypothesis. The present double replica electroblotting method was successfully applied to the combined phenotyping of C6 and C7 from bloodstains which were stored at room temperature for up to 4 weeks. The method is quite suitable for medicolegal examination of bloodstains particularly for the saving in the amount of sample.

alpha-L-fucosidase phenotyping in human tissues, dental pulps and hair roots.

Phenotypes of alpha-L-fucosidase (Fu) were demonstrated from tissue samples of spleen, liver, lung and kidney stored for a few weeks at room temperature. Activity was fairly low in pancreas, heart, muscle, skin and brain, and typing was not reliable after 1 week storage. Fu types were detectable from dental pulp tissue of up to 1 week storage. Activity was present in hair root cells, but was extremely low. The results show that the Fu typing may be applicable to individual identification of organ tissues and teeth.

PGD phenotyping in bloodstains, organ tissues, dental pulps and hair roots by isoelectric focusing.

Polymorphism of PGD was investigated in bloodstains, organ tissues, dental pulps, hair roots and semen by isoelectric focusing. This technique provided much higher resolution of PGD isoenzymes than starch gel electrophoresis. Phenotyping was possible from bloodstains for 5 weeks, from organ tissues (except pancreas) for 1-3 weeks, from dental pulps for 2 weeks and from hair roots for 2 weeks when they were stored at room temperature. The method is simple, rapid, reliable and therefore useful in medicolegal individualization of bloodstains, organ tissues, teeth and hairs.

In vitro detection of equine arteritis virus from seminal plasma for identification of carrier stallions.

Equine arteritis virus (EAV) was readily isolated in RK-13 cell monolayers by plaque assay from seminal plasma of experimental carrier stallions when they contained high titers of virus regardless of the presence of non-viral cytotoxicity in the seminal plasma. The cytotoxicity interfered with virus isolation from seminal plasma which contained virus at titers less than 10 PFU/ml. However, it was possible to detect the virus in seminal plasma pretreated with PEG (#6000). EAV was consistently identified by RT-PCR from crude seminal plasma which contained virus at titers of more than 10(2.7) PFU/ml. In vitro detection of EAV by virus isolation supplemented with RT-PCR using seminal plasma was proved to be an effective alternative to the standard test mating as a diagnostic method for carrier stallions.

Simultaneous determination of FUC and PGM1 by isoelectric focusing.

This paper describes a simple procedure for the simultaneous detection of FUC and PGM1 in semen, seminal stains, dental pulps and hair roots after separation by isoelectric focusing using Ampholine pH 5-7. The gel plate was first stained for FUC using an agarose overlay with a buffer of pH 7.5. After reading the FUC types, the FUC overlay was removed and replaced with an agarose overlay for PGM. The results show that the method is useful in medicolegal individualization of seminal stains and teeth when only small samples have to be examined.

Nasal allergy in spinning mill workers and the possibility of allergenicity of chemical fibers and cotton linters.

During a three-year period from 1976, we examined fifty eight patients with nasal allergy who worked in spinning mills or handled sewing machines. Skin tests and procative nasal tests were performed on these patients with crude and fractionated extracts from cotton linters, cotton cloth and chemical fibers. The crude aqueous antigens were extracted in dextro-phenol solution and fractionated by Saphadex G-100. It was possible that the cotton and chemical fibers included components which were the cause of hypersensitivity, but there is still doubts concerning their reaction as irritant substances. Serum specific IgE antibody was found in extracts from chemical fiber. Therefore, spinning mill dust acts as an irritant of the nose add the possibility of antigenicity of the dust for immediate type nasal allergies can not be denied from the present study.

Polymorphism of EsD by isoelectric focusing: phenotyping in human tissues, dental pulps, hair roots, and semen.

The polymorphism of EsD was investigated in tissues of various human organs, dental pulps, hair roots, and seminal stains by isoelectric focusing. The method yielded an excellent resolution of the isoenzyme components. The time limits of determination were: in organ tissues 3 weeks, in dental pulps 1 week, and in hair roots several days. The 7-1 type was less stable than the common types. Phenotyping was possible from fresh semen samples, but was unsuccessful from dried seminal stains after storage. The results show that the EsD typing by isoelectric focusing is of practical use for medicolegal individualization of organs, teeth, and hairs.

DIA3 phenotyping in human tissues, dental pulps, and hair roots by isoelectric focusing.

The polymorphism of DIA3 was investigated in tissues of various human organs, dental pulps, and hair roots by isoelectric focusing. DIA3 types were demonstrated from tissues of brain, prostate, testis, ovary, and uterus, but not from tissues of spleen, pancreas, heart, liver, muscle, lung, skin, and kidney. Determination was possible from dental pulps stored at room temperature for up to 2 weeks and from fresh hair roots. The results show that the DIA3 typing by isoelectric focusing is useful for medicolegal individualization of brain, reproductive organs, teeth, and hairs.