On nitrocellulose membrane (NCM), a spot of acetylcholine droplet (ACh) was precipitated with silicotungstic acid (STA). NCM was incubated either with rabbit polyclonal antiserum against ACh or with rabbit non-immune serum. When visualized by an immunostaining procedure using goat anti-rabbit IgG and rabbit peroxidase-anti-peroxydase (PAP) complex, both sera gave similar positive reaction products on the spot. The second antibody (goat anti-rabbit IgG) alone, namely omitting the first antibody, also gave positive immunoperoxidase reaction. The application of PAP alone gave faint but similarly positive staining. The binding of IgG molecules appears to be concerned with the ACh moiety, but not with STA moiety of the precipitate of acetylcholine silicotungstate (ACh-STA) complex. We thus consider this phenomenon as avidity of IgG for ACh. Other quaternary ammonium compounds such as decamethonium, eserine, d-tubocurarine and atropine are also targets of IgG avidity. The solution of ACh at 10-5 M facilitated the avidity, while 10-2 to 1 M ACh inhibited the avidity. Purified rabbit IgG and Fab revealed a distinct avidity, while Fc had only a weak avidity. The IgG avidity for ACh was discussed in relation to two biological phenomena: autoimmunity against ACh of the experimental animals and structural similarity of IgG molecule with nicotinic ACh receptor.
The immobilization of cell-adhesive proteins onto titanium implant improves biological responses at the implant-tissue interface. The aim of this study is to investigate how the reaction temperature during tresylation and pretreatment of titanium surface with H2O2 solution influences the reactivity of titanium basic terminal OH group towards 2,2,2-trifluoroethanesulfony1 chloride (tresy1 chloride) and fibronectin. Three different titanium surfaces were prepared, i.e., mirror polished, and pretreated either with 0.1 M H2O2/phosphate-buffered saline (PBS) solution (H2O2/PBS) or with 0.1 M H2O2/0.1 M NaOH aqueous solution (H2O2/NaOH) after mirror polishing. The basic terminal OH groups of titanium were allowed to react with tresyl chloride at 37°C or 70°C for 2 days. The tresylated titanium disk was then immersed into a fibronectin/PBS solution. The reactivity of titanium basic terminal OH group towards tresyl chloride and fibronectin was evaluated by the measurement of X-ray photoelectron spectroscopy (XPS). The reaction of titanium basic terminal OH group towards tresyl chloride at 37°C proceeded more effectively than that at 70°C. The basic OH group on H2O2/PBS-pretreated titanium showed the highest reactivity towards tresyl chloride by evaluating the F1s intensities in XPS measurement. Fibronectin could easily attach the tresylated titanium, and the attached fibronectin remained after 60 min ultrasonic cleaning regardless the different surfaces. Therefore, we concluded that after pretreatment with 0.1 M H2O2/PBS, reaction of titanium with tresyl chloride should be performed at 37°C in order to get easy attachment of fibronectin on titanium surface.
T helper (Th) cells have been characterized into two subsets, Th1 and Th2, by the pattern of cytokine secretion. Generally, Th1 type cells mediate cellular immune responses and Th2 type cells me diate humeral immunity. Recently, Th1 type immunity is thought to be necessary to exclude hepatitis B virus (HBV). Although enhanced expression of Th1 type cytokine is found in patients after interferon-alpha (IFN-α) treatment, the mechanism of this therapeutic effect has not been clarified adequately. In this study we investigated the effects of several IFN-α subtypes on the balance between Th1 and Th2 cells in eleven patients with HBV, by stimulating their peripheral blood mononuclear cells (PBMC) with phorbol 12-myristate 13-acetate (PMA) and ionomycin. Cytokine producing cells were determined by flow cytometty after 48 h of stimulation with several IFN-α subtypes. Compared with the control, IFN-α2, IFN-α8 and lFN-α10 exhibited up-regulation of the Th1/Th2 ratio by both increasing the Th1 cell population and/or decreasing the Th2 cell population. IFN-α8 was the most potent cytokine for changing the Th1/Th2 balance among the IFN-α subtypes tested. IFN-α5, however, revealed an inverse effect and IFN-α1 did not show any significant changes. These findings imply that IFN-α subtypes modify the Th1/Th2 balance in PBMC from patients with HBV, thus suggesting a useful form of IFN therapy for the HBV-infected patients that have an imbalance between Th1 and Th2 cell populations. Furthermore, our findings should help to elucidate the mechanism underlying IFN therapy for patients with HBV infection.
Calcium phosphate (Ca-P) ceramics are well known for their excellent osteo-conductivity. However, factors critical to the mineralization process of Ca-P ceramics are not fully understood, including the importance of the crystal system, the Ca/P ratio, the solubility and the surface morphology of the materials. Therefore, this study was performed to evaluate the initial mineralization behavior of osteoblast-like cells on Ca-P ceramics of different crystal systems and Ca/P ratios but with similar solubilities and surface morphologies. Cell attachment and cell proliferation were not significantly different among hydroxyapatite, β-tricalcium phosphate and titanium. However, the activity of alkaline phosphatase, the concentration of osteocalcin and the formation of mineralized nodules revealed that the initial mineralization of osteoblast-like cells was better on Ca-P ceramics than on titanium, although no significant differences were found between β-tricalcium phosphate and hyclroxyapatite. In conclusion, this study demonstrates that differences in the crystal system are not a predominant factor in the initial mineralization process of osteoblast-like cells, suggesting that the solubility of Ca-P ceramics may play an important role in the clinical implant treatment.
The distribution and type of iminunocompetent cells were investigated in rat oral mucosae using immunocytochemistry and enzyme histochemistry, focusing on histological structures. We used two antibodies, OX6 and ED1, which recognize the rat Ia-antigen and macrophage/monocyte lineage, respectively. Enzymatic histochemistry for acid phosphatase (ACPase) activity and adenosine triphosphatase (ATPase) activity was also employed to identify macrophages and Langerhans cells, respectively. Many OX6-immunopositive cells, dendritic or irregular in shape, were recognizable in the lamina propria of oral mucosae: some cells extended their dendritic processes into the epithelial layer of the buccal and sublingual mucosae. Dendritic cells within the epithelium showed intense ATPase reaction, indicating they could be categorized as Langerhans cells. A small number of ED1-positive cells existed in the lamina propria, but none were present in the epithelial cell layer. Double staining either with OX6 and ED1 or OX6 and ACPase made it possible to divide the immunocompetent cells in the lamina propria into three types: the OX6-positive cells without ED1 or ACPase-reaction, the OX6-negative cells with ED1 and ACPase-reactions, and the OX6-ED1/ACPase-co-expressing cells, each of which possessed characteristic ultrastructural features demonstrated by immunoelectron microscopy. Taking these findings together with previous reports, these three types of cells were regarded as a dendritic-like cell, a macrophage without antigen-presentation ability, and a macrophage with antigen-presentation ability, respectively. There were regional differences in the distribution and density of these iminunocompetent cells; they were densely distributed in order of the buccal and sublingual nrucosae, the palatal mucosa, and the dorsal surface of tongue. The region-specific distribution and density of the immunocompe tent cells might be due to the histological structure of each oral mucosa, suggesting the presence of different immune-defense systems among each portion of the oral mucosae.
The number of surgically resected cases of extrahepatic bile duct cancers has been increased because of the recent advances in imaging diagnoses and operative procedures. However, morphological characteristics of the extrahepatic bile duct cancers have not yet been defined. In this study, we reviewed 102 surgically resected cancer cases for clarifying growth and invasion of the extrahepatic bile duct cancer. We designated as “early cancer”cancers limited to the mucosa or invading as far as the fibromuscular layer (smooth muscle layer), i.e., Tis or T1 cancers. Approximately 12.7% (13/102) of the cases were pTis or pT1 cancers (pathologic stage of Tis or T1), which were categorized as early cancer. Only 15.4% (2/13) of the early cancers showed vascular/perineural invasion and/or lymph node metastasis. In contrast, the majority (87.3%) of the cases was pT2-3 cancers, and very frequently showed vascular/perineural invasion and/or lymph node metastasis. Desmin immunostaining was very useful for recognizing thin muscle layer, and should be performed for pathological diagnosis of early extrahepatic bile duct cancers.
The cDNAs coding for two distinct types of aspartic proteases, Bm-ASP-1 and Bm—ASP-2, were cloned from the parasitic nematode Brugia malayi, an important pathogen of human lymphatic filariasis, and their amino acid sequences (393 and 452 residues, respectively, as prepro forms) were deduced. Bm-ASP-1 and Bm-ASP-2 are 29% identical with each other in amino acid sequence and phylogenetic analysis indicated that Bm-ASP-1 and Bm-ASP-2 belong to two different subfamilies of aspartic proteases of nematodes. lmmunohistochemical analysis indicated that Bm-ASP-1 is localized in the intestine and esophagus, while Bm-ASP-2 is distributed more ubiquitously in various tissues including intestine, esophagus, body wall muscle and reproductive system, suggesting their different roles in vivo.