Nearly one hundred years ago, acetylcholine (ACh) was proposed as a chemical agent responsible for nerve transmission at the synapse, the junction area between one neuron and its target cell. Since it has been proved that ACh played, indeed, a major role in the functioning of the nerve system in the vertebrates, cholinergic nerve transmission became a basic field of study in neuroscience. The birth of histochemistry and its ulterior developments allowed in situ localization of the molecular agents related to the functioning of the cholinergic synapse. This report presents historical landmarks in the histochemistry of major cholinergic agents (acetylcholinesterase, nicotinic acetylcholine receptor, choline acetyltransferase, and ACh), a domain which has greatly contributed to the knowledge of the nerve system. It is emphasized that despite extraordinary progresses made in this field, basic problems, such as in situ localization of ACh, still remain to be solved.
The invasion of intraductal papillary-mucinous neoplasm (IPMN) is sometimes difficult to diagnose using only ordinary hematoxylin-eosin sections. The aim of this study was to evaluate the invasion of IPMN more precisely using thrombospondin-1 (TSP1) immunohistochemistry as a useful adjunct to morphological examination. Eighty patients that underwent primary resection for pancreatic IPMNs were retrospectively analyzed. The 80 IPMNs were studied for the expression of TSP1, MUC1-CORE, MUC2, and MUC5AC. The cases were evaluated for dysplasia, the presence of invasion, hisological subtypes, and survival. The 80 IPMNs were classified into 29 intraductal papillary-mucinous adenomas (IPMAs), 10 borderline IPMNs, 18 noninvasive intraductal papillary-mucinous carcinomas (IPMCs), and 23 invasive IPMCs according to the WHO classification. Invasive IPMCs were further divided into 12 minimally invasive IPMCs (MI-IPMCs) and 11 invasive carcinomas originating from IPMCs (IC-IPMCs) according to the Japan Pancreatic Society classification. The rate of strongly positive cases with more than 30% of the cancer stroma area expressing TSP1 was significantly higher in MI-IPMC and IC-IPMC than in noninvasive IPMC (P = 0.035, 0.005). Furthermore, patients in the strongly positive group had a significantly poorer prognosis compared to patients in the negative-weakly positive group (P = 0.008, log-rank test). Of the 80 tumors, 22 were classified into gastric-, 45 into intestinal-, 7 into pancreatobiliary-, and 6 into oncocytic-type IPMNs according to criteria described previously. The cases with a strongly positive expression of TSP1 were frequently detected in the pancreatobiliary and oncocytic types (P = 0.001). In conclusion, stromal TSP1 expression is a prognostic indicator and a new marker of invasiveness in IPMN.
Despite the existence of several methods for the diagnosis of oral infectious diseases, few rapid and quantitative methods exist for discriminating between live and dead bacterial cells in oral clinical samples. In this study, we characterized a light-emitting diode (LED) fluorescence microscopic technique for quantifying live and dead oral bacterial cells stained with 4',6'-deamidino-2-phenyllindole and propidium iodide. Four bacterial strains representative of the human oral microflora were used in this study. In addition, saliva and subgingival fluid specimens were collected from healthy volunteers. Saliva was obtained from the donors without stimulation, whereas subgingival fluid was obtained by inserting a sterile endodontic paper point into the subgingival sites of the first molar. The samples were cultured on agar plates and subjected to LED microscopy. The correlations between both methods were analyzed. The number of live bacterial cells as determined by LED-based fluorescence microscopy and standard colony counts on agar plates correlated well for the known oral bacterial strains and bacterial cells in the clinical specimens. The LED illumination method characterized in this study can be used for the rapid enumeration of living and dead cells. However, to show specificity, this method requires further innovations.
Adipose-derived stem cells (ADSCs) and their secretomes mediate diverse skin-regeneration effects, such as wound-healing and antioxidant protection, that are enhanced by hypoxia. We investigated the hair-growth-promoting effect of conditioned medium (CM) of ADSCs to determine if ADSCs and their secretomes regenerate hair and if hypoxia enhances hair regeneration. If so, we wanted to identify the factors responsible for hypoxia-enhanced hair-regeneration. We found that ADSC-CM administrated subcutaneously induced the anagen phase and increased hair regeneration in C3H/NeH mice. In addition, ADSC-CM increased the proliferation of human follicle dermal papilla cells (HFDPCs) and human epithelial keratinocytes (HEKs), which are derived from two major cell types present in hair follicles. We investigated the effect of hypoxia on ADSC function using the same animal model in which hypoxia increased hair regrowth. Forty-one growth factors in ADSC-CM from cells cultured under hypoxic or normoxic conditions were analyzed. The secretion of insulin-like growth factor binding protein (IGFBP)-1, IGFBP-2, macrophage colony-stimulating factor (M-CSF), M-CSF receptor, platelet-derived growth factor receptor-β, and vascular endothelial growth factor was significantly increased by hypoxia, while the secretion of epithelial growth factor production was decreased. It is reasonable to conclude that ADSCs promote hair growth via a paracrine mechanism that is enhanced by hypoxia.
This aim of this study was to determine the characteristic differences in tendinocytes derived from three sites of the equine superficial digital flexor tendon (SDFT)—proximally the myotendinous junction (MTJ), mid-metacarpal (mM) and osteotendinous junction (OTJ)—in morphology, proliferation, and ability for synthesis of collagen and matrix metalloproteinases (MMPs). Little difference was observed in cell proliferation. Addition of tumor necrosis factor (TNF) α to the culture medium resulted in increased collagen synthesis by tendinocytes from all three sites. The amount of collagen synthesized by tendinocytes derived from the mM and OTJ was much larger than that synthesized by untreated tendinocytes. A collagen zymogram revealed that proMMP-13 synthesis was increased towards the distal site. However, TNFα treatment resulted in a significant decrease in the amount of proMMP-13 synthesized by tendinocytes from all three sites. On the other hand, a gelatin zymogram showed that the synthesis level of proMMP-9 tended to decrease towards the distal site, but there was little difference between synthesis levels of proMMP-9 before and after TNFα treatment. These results indicated that tendinocytes in the same tendon have different characteristics and that these characterisities would reflect the function of tendinocytes in vivo. Also, the isolated tendinocytes provided much information on the characteristics and properties of tendons for the ECM turnover system and on the responsiveness of tendinocytes to complex inflammatory responses in a tendinopathy condition.
4-Methylcatechol (4MC), a stimulator of the synthesis of neurotrophin family members in various cells, was able to up-regulate the expression of heme oxygenase (HO)-1, a redox-sensitive inducible stress protein, in neural stem/progenitor cells (NS/PCs). RT-PCR analysis showed that 4MC induced HO-1 mRNA expression in a dose- and a time-dependent manner. The increase in HO-1 mRNA was followed by an increase in HO-1 protein content, which was confirmed by ELISA and Western blotting analysis. When NS/PCs were pretreated with 4MC before exposure to hydrogen peroxide (H2O2), most of the cells were rescued from the H2O2-induced death. 4MC enhanced the phosphorylation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) and Akt in a time-dependent manner. Pretreatment of cultures with a selective inhibitor of PI3 kinase (PI3K)/Akt, but not with one of MAPK/ERK, inhibited both the 4MCinduced HO-1 expression and neuroprotective effect, demonstrating that PI3K/Akt signaling pathway played a significant role in 4MC-induced HO-1 induction and neuroprotection. Taken together, our results suggest that 4MC activates the expression of HO-1 through the PI3K/Akt signaling pathway and that the HO-1 protein inhibits the death of NS/PCs induced by oxidative stress.
Human T cell leukemia virus type I (HTLV-I), a causative agent of adult T-cell leukemia (ATL), is transmitted from mother to child predominantly by breastfeeding. The source of HTLV-I-infected cells in breast milk has been thought to be T cells, however, the majority of cells in breast milk are CD14+ macrophages but not CD3+ T lymphocytes, and no data are available regarding HTLV-I transmission through breast milk macrophages (BrMMφ). To explore the potential of BrMMφ as a possible source of infection in mother to child transmission (MTCT) of HTLV-I, an immortalized cell line (HTLV-BrMMφ) has been established from BrMMφ by infection with HTLV-I. HTLV-BrMMφ retained macrophage characteristics and did not express a complete dendritic cell (DC) phenotype; nevertheless, HTLV-BrMMφ efficiently promoted T cell proliferation in primary allogeneic mixed lymphocyte reaction (MLR) like DC. Moreover, HTLV-I infection could be transmitted from HTLV-BrMMφ to activated T cells in the peripheral blood. These findings suggested that BrMMφ might be an appropriate HTLV-I reservoir involved in MTCT transmission via breastfeeding.
High molecular weight hyaluronan (HA) is widely used in the treatment of osteoarthritis (OA) and rheumatoid arthritis (RA) by intra-articular injection. However, comparative studies of HA actions on catalytically activated cartilages in different pathologic conditions have rarely been investigated. This study was aimed to compare the inhibitory effects of HA on nitric oxide (NO) production by COOH-terminal heparin-binding fibronectin fragment (HBFN-f) between normal and diseased cartilages. When articular cartilage explants from normal, OA, or RA joints were incubated with HBFN-f, the RA and OA cartilages produced higher levels of NO compared with normal cartilage. Pretreatment with 2700 kDa HA resulted in significant suppression of HBFN-f-stimulated NO production in OA and RA cartilages. While CD44 was up-regulated in OA and RA cartilages, anti-CD44 antibody reversed HA inhibition of HBFN-f action in those cartilages. The present results clearly demonstrated that HA blocked HBFN-f actions in OA and RA cartilages through interaction with CD44. HA, which targets CD44 highly expressed on OA and RA chondrocytes, could suppress catabolic actions by fibronectin fragments like HBFN-f in diseased cartilage.
To examine the molecular diversity of the venom proteins of the Russell's viper (Daboia russellii russellii) and the Indian cobra (Naja naja) in Sri Lanka, we isolated 38 venom proteins through a combination of anion exchange chromatography followed by reversed-phase high performance liquid chromatography. From the venom of D. r. russellii we isolated 15 proteins: 5 isozymes of phospholipase A2 (PLA2), 4 serine proteases, 2 C-type lectin-like proteins, 2 L-amino acid oxidases, 1 cysteine-rich secretory protein (CRISP), and 1 metalloproteinase. From the venom of N. naja we isolated 23 proteins: 10 isoforms of cytotoxins (CTX), 7 PLA2 isozymes, 2 muscarinic toxinlike proteins, 2 CRISPs, 1 nerve growth factor, and 1 new thrombin-like serine protease. Most of these proteins contained new amino acid sequences for each species, indicating molecular diversity in venom proteins. The entire amino acid sequences of PLA23 from D. r. russellii and CTX7 from N. naja were determined. Additionally, the polymorphic amino acid residues of PLA23 were preferentially localized on the potential antigenic sites. While 2 types of PLA2 (N and S types) were found in D. r. russellii (India) and D. r. siamensis (Java), all the PLA2s from D. r. siamensis (Burma) were N type, and those from D. r. russellii (Sri Lanka) were primarily S type.
The LEC rat has a spontaneous mutation of T-helper immunodeficiency (thid), which causes a marked defect in T cell maturation from double positive (DP) to CD4 single positive (SP) cells in the thymus. Previously, we identified the contiguous gene deletion of Ptprk and Themis genes in the thid locus that causes the simultaneous loss of expression of both genes, and the exogenous Ptprk expression partially rescued this phenotype. To determine whether the deletion of Themis influences thid phenotype, bone marrow (BM) cells were transduced with lentiviral vector expressing Themis gene, and were transplanted into X-ray-irradiated LEC rats. Interestingly, the exogenous Themis expression rescued the development of CD4 SP cells as well as Ptprk. The result suggests that the deficiency of both genes is responsible for the thid mutation, and that both genes are indispensable for the development of SP cells from DP cells in the thymus.