Expression of DOK1, 2, and 3 genes in HTLV-1-infected T cells.

Human T-cell leukemia virus type 1 (HTLV-1) can cause an aggressive malignancy known as adult T-cell leukemia/lymphoma (ATLL). The Tax protein encoded by the pX region of the HTLV-1 genome appears to be a key element in the early stage of ATLL development. In this study, we examined the expression of the downstream of tyrosine kinase (DOK) family members DOK1, DOK2 and DOK3, recently reported to be tumor suppressors, in HTLV-1-transformed T cells (MT-2 and HUT-102) and TL-Om1 cells derived from ATLL leukemic cells. DOK2 and DOK3 expression was significantly reduced in MT-2, HUT-102, and TL-Om1 cells compared with their expression in uninfected T cells, and the expression of DOK3 was reduced by the induction of Tax expression in T cells.

Cyclophosphamide eliminates suppressor T cells in age-associated central regulation of delayed hypersensitivity in mice.

Effect of treatment of mice with cyclophosphamide (CY) on the delayed hypersensitivity (DH) response was investigated in C57BL/6 mice. DH to methylated human serum albumin (MHSA) could be enhanced with CY in young mice but not in aged ones. DH enhancement with CY appeared to be due to elimination of suppressor T cells involved in DH. Effector T cells were also sensitive to CY, the damaging effect of CY on these latter cells was, however, transient suggesting the rapid recovery of effector T cells. The overshooting recovery of the effector T cells required the presence of the thymus. It is more probably that there are at least two distinct subpopulations of T cells in DH, effector T cells, and suppressor T cells. The distinction is already apparent in the thymus stage. The suppressor T cells, categorized as a central regulator, seem to be antigen nonspecific and regulate the more effectively the DH in young mice, thus physiological role of these cells in age-associated immune alterations is implicated.

Studies on delayed hypersensitivity in mice. III. Evidence for suppressive regulatory T1-cell population in delayed hypersensitivity.

T-T-cell interactions involved in delayed hypersensitivity (DH) response have been studied by employing delayed foot pad assay to methylated human serum albumin in C57BL/6J mice. The DH response, one of the T-cell manifestations of cell-mediated immune response is suppressively regulated by T cells and such observation was based on studies of age-associated kinetics of foot pad reaction and effects of cell transfer and adult thymectomy on developing DH response. These suppressively regulatory T cells in DH have a life span of less than 4 wk and a constant derivation from the thymus is required. Such cells are numerous in the young mouse thymus and few in the spleen and thymus of old mice. On the one hand, the presence of a long-lived effector T-cell population was suggested in DH. These cells are numerous in the spleen and are low responders to phytohemagglutinin in vitro. It is assumed that these suppressive T cells interact with antigen-reactive cells at their proliferating stage by recognition of the iodiotypic difference through surface receptors. As in the case of graft-vs.-host and humoral response in vivo, three different subsets of immune competent cells participate in the DH response. These cells consist of one specifically antigen-reactive T cell, one suppressive regulatory T cell, and one bone marrow-derived cell, a macrophage that responds to a chemical mediator from sensitized effector T cells and that develops a DH skin lesion nonspecifically.

Assessment of the potential for pathogen dispersal during high-flow nasal therapy.

High-flow nasal therapy is increasingly used in hospitals because of its effectiveness and patient comfort. However, pathogens in the patient's nasal and oral cavities may be dispersed by forced air. This study aimed to investigate the risk of pathogen dispersal during high-flow nasal therapy. Liquid and bacterial dispersal were assessed via in-vitro experimental set-ups using a manikin. Thickened water or fresh yeast solution mimicked saliva and nasal mucus secretions. Dispersal was limited to the proximal area of the face and nasal cannula, suggesting that high-flow nasal therapy does not increase the risk of droplet and contact infection.

Antigen-binding cells in normal mouse thymus.

The thymus of a normal adult mouse contains lymphocytic cells with a large number of antigen-binding receptors. Binding of the antigen by these cells is specific and can be inhibited by cross-reactive materials. It is possible that the interaction of thymocytes with antibody-forming precursor cells, required in the primary immune response to certain antigens, is mediated by these specific antigen-binding cells of the thymus.

Recurrent Spindle Cell Carcinoma Shows Features of Mesenchymal Stem Cells.

This study investigated a case of spindle cell carcinoma (SpCC) in tongue pathological lesions. The patient experienced a local recurrence and distant metastasis after surgical intervention. Although standard chemotherapy was administered, a granulomatous mass continued to develop. This aggressive growth led to survival of the tumor. Secondary debulking surgery was performed to improve the patient's quality of life at the request of the patient. Using a tissue sample derived from the secondary debulking surgery, we performed an analysis of the tumor's cell surface antigens, differentiation potential, metastatic ability, and inhibition potential by anticancer reagents. In vitro analysis revealed that the cell population grown under adherent culture conditions expressed the mesenchymal stem cell (MSC) markers CD73, CD90, and CD105. The cell line established from this SpCC contained colony-forming unit fibroblasts (CFU-Fs) and exhibited multipotent differentiation into several mesenchymal lineages, including bone, cartilage, and fat. The SpCC cells also displayed vigorous mobilization. These characteristics suggested that they had the differentiation potential of mesenchymal cells, especially MSCs, rather than that of epithelial cells. The surgical specimen analyzed in this study resisted the molecular target reagent cetuximab, which is an epidermal growth factor receptor inhibitor. This clinical insight revealed that chemotherapy-resistant SpCC cells have different characteristics compared to most other cancer cells, which are sensitive to cetuximab. Our cell death assay revealed that SpCC cell death was induced by the anticancer drug imatinib, which is known to inhibit protein tyrosine kinase activity of ABL, platelet-derived growth factor receptor α (PDGFRα), and KIT. Here, we report recurrent SpCC with characteristics of MSCs and potential for treatment with imatinib.

Ablation of neuropsin-neuregulin 1 signaling imbalances ErbB4 inhibitory networks and disrupts hippocampal gamma oscillation.

Parvalbumin-expressing interneurons are pivotal for the processing of information in healthy brain, whereas the coordination of these functions is seriously disrupted in diseased brain. How these interneurons in the hippocampus participate in pathological functions remains unclear. We previously reported that neuregulin 1 (NRG1)-ErbB4 signaling, which is actuated by neuropsin, is important for coordinating brain plasticity. Neuropsin cleaves mature NRG1 (bound to extracellular glycosaminoglycans) in response to long-term potentiation or depression, liberating a soluble ligand that activates its receptor, ErbB4. Here, we show in mice that kainate-induced status epilepticus transiently elevates the proteolytic activity of neuropsin and stimulates cFos expression with a time course suggesting that activation of ErbB4- and parvalbumin-expressing interneurons follows the excitation and subsequent silencing of pyramidal neurons. In neuropsin-deficient mice, kainate administration impaired signaling and disrupted the neuronal excitation-inhibition balance (E/I balance) in hippocampal networks, by decreasing the activity of parvalbumin-positive interneurons while increasing that of pyramidal neurons, resulting in the progression of status epilepticus. Slow, but not fast, gamma oscillations in neuropsin-deficient mice showed reduced power. Intracerebroventricular infusion of the soluble NRG1 ligand moiety restored the E/I balance, status epilepticus and gamma oscillations to normal levels. These results suggest that the neuropsin-NRG1 signaling system has a role in pathological processes underlying temporal lobe epilepsy by regulating the activity of parvalbumin-expressing interneurons, and that neuropsin regulates E/I balance and gamma oscillations through NRG1-ErbB4 signaling toward parvalbumin-expressing interneurons. This neuronal system may be a useful target of pharmacological therapies against cognitive disorders.

Structural comparison of the free and DNA-bound forms of the purine repressor DNA-binding domain.

BACKGROUND: The purine repressor (PurR) regulates genes that encode enzymes for purine biosynthesis. PurR has a two domain structure with an N-terminal DNA-binding domain (DBD) and a C-terminal corepressor-binding domain (CBD). The three dimensional structure of a ternary complex of PurR bound to both corepressor and a specific DNA sequence has recently been determined by X-ray crystallography. RESULTS: We have determined the solution structure of the PurR DBD by NMR. It contains three helices, with the first and second helices forming a helix-turn-helix motif. The tertiary structure of the three helices is very similar to that of the corresponding region in the ternary complex. The structure of the hinge helical region, however, which makes specific base contacts in the minor groove of DNA, is disordered in the DNA-free form. CONCLUSION: The stable formation of PurR hinge helices requires PurR dimerization, which brings the hinge regions proximal to each other. The dimerization of the hinge helices is likely to be controlled by the CBD dimerization interface, but is induced by specific-DNA binding.

Spontaneous occurrence of atypical hyperplasia and adenocarcinoma of the uterus in androgen-sterilized SD rats.

In SD female rats sterilized by a single injection of testosterone propionate at 2 days after birth, the spontaneous occurrence of atypical hyperplasia and adenocarcinoma of the uterus was observed for a fairly long period (greater than 2 yr). Two atypical hyperplasias and 2 adenocarcinomas were detected in 25 androgen-sterilized rats (ASR) after 500 days of age; in contrast, in 111 normal control rats no abnormal uterine proliferation was detected during a 750-day observation period. These results indicate that a persistence of both hormone imbalances and dysfunctional uteri in ASR induces abnormal uterine proliferation at a late age.

Effect of 1 alpha,25-dihydroxyvitamin D3 on proliferation of activated T-cells and established human lymphotropic virus type I-positive T-cell lines.

The effects of 1 alpha, 25-dihydroxyvitamin D3 [1,25-(OH)2D3] on proliferation and de novo DNA synthesis were studied in the following established human leukemia cell lines: lymphoblastic T-cell lines HPB-ALL, CCRF-HSB-2, p12/lchikawa, and HPB-MLT; adult T-cell leukemia- (ATL) and human lymphotropic virus type I (HTLV-I)-infected T-cell lines HUT102, HUT-102B2, MT-1, MT-2, MJ, C2/MJ, KH-2, KH-2Lo, HPB-CTL-1, and ATN-C1; ATL-derived B-cell lines ATL-BK9 and ATL-BK10; lymphoblastic B-cell line Daudi; and myelocytic-monocytic lineage cell lines HL-60 and U937. 1,25(OH)2D3 inhibited proliferation and de novo DNA synthesis of phytohemagglutinin-P-activated T-cells and certain established HTLV-I-positive T-cells. However, it did not inhibit immature lymphoblastic T- and B-cells or ATL-derived B-cells. The degree of inhibition depended on the dose of 1,25(OH)2D3 and the heterogeneity of the established HTLV-I-positive T-cells. KH-2 and subclone KH-2Lo were markedly inhibited, and HPB-CTL-1 was moderately inhibited. Marked inhibition of DNA synthesis in KH-2Lo cells was observed in the proliferative phase of the cell cycle. No inhibition of KH-2Lo proliferation or expression of interleukin-2 and transferrin receptor was noted after removal of 1,25(OH)2D3 from the culture medium. 1,25(OH)2D3 inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced multinucleated cell formation of various HTLV-I-positive T-cell lines and TPA-induced HTLV-I p19 expression in KH-2Lo cells.

Antitumor activity of D-mannosamine in vitro: different sensitivities among human leukemia cell lines possessing T-cell properties.

D-Mannosamine is toxic to human malignant T-lymphoid cell lines derived from patients with T-cell leukemia. We observed heterogeneity of mannosamine susceptibility among those cell lines. The leukemic T-cell lines, subgrouped according to the degree of mannosamine inhibition on nucleic acid biosyntheses, were: Subgroup 1, HPB-MLT cells; Subgroup 2, CCRF-HSB-2 and HPB-ALL cells; and Subgroup 3, MOLT-4 cells. The most sensitive line, HPB-MLT, originated from the patient with adult T-cell leukemia. The cytotoxicity of mannosamine was potentiated by a fatty acid, sodium oleate, at concentrations that were noncytolytic, and the interaction between the two drugs was synergistic. These results would suggest that mannosamine induces changes in the membrane structure of the leukemia cells. Thus, the primary target of the tumoricidal activity of mannosamine may also be the cellular membranes.

Phorbol ester-induced differentiation of human T-lymphoblastic cell line HPB-ALL.

12-O-Tetradecanoylphorbol-13-acetate (TPA), a potent tumor promoter, induced phenotypic differentiation in the human thymic acute lymphocytic leukemia cell line, HPB-ALL. Within 30 min of seeding in the presence of TPA, the cells formed a smooth round shape. After a 7-day exposure to TPA, most of the cells became smaller and reminiscent of large or atypical lymphocytes. Electron microscopic analysis evidenced morphological differentiation in TPA-treated HPB-ALL cells. Thymic antigens stained with monoclonal antibody OKT6 were dramatically reduced while Leu2a-positive cells were increased in the TPA-treated HPB-ALL cells. However, OKT3-positive cells did not appear in these TPA-treated cells for up to 7 days. Upon TPA-induced phenotypic differentiation, the growth rate of cells was significantly inhibited, their ability to incorporate DNA and RNA via 3H-labeled precursors was reduced, their ability to bind sheep red blood cell rosettes was significantly increased, and the proportion of terminal deoxynucleotidyl transferase-positive cells was decreased.

Human T-cell leukemia virus type I tax activates transcription of the human monocyte chemoattractant protein-1 gene through two nuclear factor-kappaB sites.

Infection by human T-cell leukemia virus type (HTLV) I leads to adult T-cell leukemia and is also associated with the neurodegenerative disease HTLV-I-associated myelopathy/tropical spastic paraparesis. Leukocytes are attracted to sites of inflammation by chemokines. One such chemokine is monocyte chemoattractant protein (MCP)-1, a member of the C-C subfamily of chemokines. We investigated whether HTLV-I infection causes up-regulation of MCP-1, which may in turn cause recruitment of leukocytes to HTLV-I-infected areas. We now report that MCP-1 mRNA levels are elevated in HTLV-I-infected T-cell lines, when compared with uninfected ones. We further confirmed secretion of MCP-1 by HTLV-I-infected T-cell lines. MCP-1 mRNA was also expressed in leukemic cells from patients with adult T-cell leukemia. The 5' transcriptional regulatory region of the MCP-1 gene was activated by the HTLV-I-encoded transactivator Tax in the human T-cell line Jurkat, in which endogenous MCP-1 is induced by Tax. By using site-specific point mutations, we have identified two closely spaced nuclear factor (NF)-kappaB sites, A1 and A2, to be important for Tax-mediated transactivation of the MCP-1 gene. Through the use of an electrophoretic mobility shift assay, we demonstrated that Tax induced NF-kappaB binding to both MCP-1 kappaB sites. This is the first report to demonstrate that Tax can transactivate the MCP-1 gene through the induction of NF-kappaB. Our results thus reveal how Tax disrupts the normally regulated MCP-1 gene and leads to its constitutive expression in HTLV-I-infected cells. These findings may have important implications for our understanding of HTLV-I-associated diseases.

Purified Human Dental Pulp Stem Cells Promote Osteogenic Regeneration.

Human dental pulp stem/progenitor cells (hDPSCs) are attractive candidates for regenerative therapy because they can be easily expanded to generate colony-forming unit-fibroblasts (CFU-Fs) on plastic and the large cell numbers required for transplantation. However, isolation based on adherence to plastic inevitably changes the surface marker expression and biological properties of the cells. Consequently, little is currently known about the original phenotypes of tissue precursor cells that give rise to plastic-adherent CFU-Fs. To better understand the in vivo functions and translational therapeutic potential of hDPSCs and other stem cells, selective cell markers must be identified in the progenitor cells. Here, we identified a dental pulp tissue-specific cell population based on the expression profiles of 2 cell-surface markers LNGFR (CD271) and THY-1 (CD90). Prospectively isolated, dental pulp-derived LNGFR(Low+)THY-1(High+) cells represent a highly enriched population of clonogenic cells--notably, the isolated cells exhibited long-term proliferation and multilineage differentiation potential in vitro. The cells also expressed known mesenchymal cell markers and promoted new bone formation to heal critical-size calvarial defects in vivo. These findings suggest that LNGFR(Low+)THY-1(High+) dental pulp-derived cells provide an excellent source of material for bone regenerative strategies.

Chemical assessment of phospholipid and phosphoenergetic metabolites in regenerating rat liver measured by in vivo and in vitro 31P-NMR.

For the assessment of 31P-NMR spectroscopic data, phospholipid precursors (phosphorylethanolamine (PE) and phosphocholine) and catabolites (glycerophosphorylethanolamine (GPE) and glycerophosphorylcholine (GPC)), as well as adenosine phosphates were chemically determined in regenerating rat liver. The data were compared with those obtained by in vivo and in vitro 31P-NMR spectroscopies. Chemical assay revealed a significant increase of PE and a decrease of GPE, GPC and ATP in hepatectomy group compared to sham operation group. The values obtained by in vitro NMR were in good agreements with those of chemical assay, but significant differences between the two groups were observed only in PE and inorganic phosphate (Pi). Noticeable increase in PME was not detected by in vivo 31P-NMR spectroscopy, although the increase of PE was about 2.5-times that of the control and its constitution ratio to the whole phosphomonoester (PME) was less than 15%. On the other hand, in vivo NMR showed a large phosphodiester (PDE) peak occupying approx. 40% of the total phosphorus signal, while the contribution of its constituents, GPE and GPC was about 5% found by both chemical assay and in vitro NMR. The PDE peak in in vivo NMR seemed to reflect the membrane phospholipid itself rather than its catabolites. A slight decrease of phosphoenergetic level in regenerating rat-liver was commonly suggested by all three analytical methods.

Interrelationship between Ca2+-dependent phospholipid base-exchange reaction and phospholipase D-like activity in bovine retina.

Endogenous substrates (phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine) for the Ca2+-dependent base-exchange reaction were investigated using bovine retinal microsomes. The amounts of the three bases, serine, ethanolamine and choline, released from the membranes and the amount of phosphatidic acid generated in the membranes were measured in the presence of Ca2+ with or without exogenous bases. When the membranes were incubated with Ca2+ alone, the three bases were liberated into the water-soluble fractions accompanied by accumulation of phosphatidic acid, suggesting the presence of Ca2+-dependent phospholipase D-like activity. When an exogenous base was added to the reaction mixture, the liberation of the other two bases increased slightly and the formation of phosphatidic acid decreased markedly. The exogenous base also stimulated the liberation of the same base from prelabeled phospholipids. Accompanying these changes, the exogenous base was incorporated into the membrane phospholipid. With respect to pH profile, time course and metal requirements, both the base incorporation and phospholipase D-like activity were quite similar. The amount of base incorporated generally agreed with both the decreased amount of phosphatidic acid formed and the increased amount of base released. These results suggest that, beside the base-exchange reaction, phospholipase D-like activity plays an important role in Ca2+-dependent base incorporation into bovine retinal membranes.

Renal ischemia/reperfusion remotely improves myocardial energy metabolism during myocardial ischemia via adenosine receptors in rabbits: effects of "remote preconditioning".

OBJECTIVES: This study examined the changes in myocardial energy metabolism during myocardial ischemia after "remote preconditioning" and investigated the involvement of adenosine receptors in the mechanisms of this effect. BACKGROUND: Recent studies have indicated that a brief period of ischemia and reperfusion (ischemic preconditioning, PC) in a remote organ reduces myocardial infarct size (IS) protecting against subsequent sustained myocardial ischemia. However, the mechanisms of "remote PC" remain unclear. We assessed myocardial energy metabolism during sustained myocardial ischemia and reperfusion after renal PC (RPC), in comparison with that after myocardial PC (MPC) in open-chest rabbits. It has been established that adenosine receptors are involved in the mechanisms of MPC. METHODS: Rabbits that had been anesthetized with halothane were divided into six groups. The control (CNT) group underwent 40-min coronary occlusion followed by 120 min reperfusion. Before the procedure, the MPC group underwent an additional protocol of 5 min coronary artery occlusion and 20 min reperfusion, and the RPC group received a 10 min episode of renal artery occlusion and 20 min reperfusion. In additional experimental groups, 8 sulfophenyl-theophylline (SPT, 10 mg/kg), an adenosine receptor inhibitor, was intravenously injected before the 40 min myocardial ischemia (SPT, MPC + SPT and RPC + SPT groups, respectively). Myocardial levels of phosphocreatine (PCr), ATP and intracellular pH (pHi) were measured by 31P-NMR spectroscopy. RESULTS: RPC and MPC delayed the decreases in ATP levels, preserved pHi during 40-min myocardial ischemia and resulted in better recovery of ATP and PCr during 120 min reperfusion compared with the controls. SPT abolished the improvement in myocardial energy metabolism and the reduction in myocardial IS caused by MPC or RPC. Myocardial IS in the CNT (n = 8), MPC (n = 9), RPC (n = 9), SPT (n = 6), MPC + SPT (n = 8) and RPC + SPT (n = 8) groups averaged 42.8+/-3.5%, 18.2+/-1.8%*, 19.6+/-1.3%*, 44.9+/-5.0%, 35.6+/-2.7% and 34.8+/-3.6% of the area at risk (*p < 0.05 vs. CNT), respectively. CONCLUSIONS: PC in a remote organ, similar to MPC, improved myocardial energy metabolism during ischemia and reperfusion and reduced IS in vivo by an adenosine-dependent mechanism in rabbits.

DNA vaccine against hamster oral papillomavirus-associated oral cancer.

Previously we developed a carcinogenesis model involving the combination of 9,10-dimethyl-1,2-benzanthracene (DMBA) application with physical wounding of hamster lingual mucosa. The presence of a novel hamster oral papillomavirus (HOPV) was demonstrated and its genome sequenced. In the present study, this HOPV hamster model was used to test whether vaccination with the L1 gene could prevent the development of oral carcinoma. DNA plasmids encoding the L1 gene or the vector alone were injected intramuscularly into 20 vaccinated and 20 control hamsters, respectively. The lingual tips of the hamsters were painted with DMBA for 8 weeks. A portion of the lingual tips was excised, and the tips were then painted daily with DMBA until the animals were killed 13 days later. All control hamsters developed lingual carcinoma, whereas 12 of the L1-vaccinated hamsters showed no lesions. These results suggest that immunization with L1 DNA vaccines may prevent the development of papillomavirus-associated oral cancer.

Increased arterial wall stiffness limits flow volume in the lower extremities in type 2 diabetic patients.

OBJECTIVE: To document an association between arterial wall stiffness and reduced flow volume in the lower-extremity arteries of diabetic patients. RESEARCH DESIGN AND METHODS: We recruited 60 consecutive type 2 diabetic patients who had no history or symptoms of peripheral arterial disease (PAD) in the lower extremities and normal ankle/brachial systolic blood pressure index at the time of the study (non-PAD group) and 20 age-matched nondiabetic subjects (control group). We used an automatic device to measure pulse wave velocity (PWV) in the lower extremities as an index of arterial wall stiffness. At the popliteal artery, we evaluated flow volume and the resistive index as an index of arterial resistance to blood flow using gated two-dimensional cine-mode phase-contrast magnetic resonance imaging. RESULTS: Consistent with previous reports, we confirmed that the non-PAD group had an abnormally higher PWV compared with that of the control group (P < 0.001). To further demonstrate decreased flow volume and abnormal flow pattern at the popliteal artery in patients with a higher degree of arterial wall stiffness, we assigned the 60 non-PAD patients to tertiles based on their levels of PWV. In the highest group, magnetic resonance angiograms of the calf and foot arteries showed decreased intravascular signal intensity, indicating the decreased arterial inflow in those arteries. The highest group was also characterized by the lowest late diastolic and total flow volumes as well as the highest resistive index among the groups. From stepwise multiple regression analysis, PWV and autonomic function were identified as independent determinants for late diastolic flow volume (r(2) = 0.300; P < 0.001). CONCLUSIONS: Arterial wall stiffness was associated with reduced arterial flow volume in the lower extremities of diabetic patients.

Inhibitory effect of novel 1-deoxynojirimycin derivatives on HIV-1 replication.

The effect of 11 derivatives of 1-deoxynojirimycin (DNM) on the replication of HIV-1 was studied. Compared with DNM, seven of them showed remarkable inhibition of HIV-1-induced syncytium formation at significantly low concentrations which were not cytotoxic. Two derivatives were found to markedly reduce the infectious virus yields from cell lines chronically infected with HIV. Analysis of HIV-1 envelope glycoproteins showed that the derivatives induced modification of the processing of not only gp120/160 but also the transmembrane glycoprotein gp41. The modification of the processing of the transmembrane glycoprotein gp41 might play an important role in the inhibition of virus replication at a step after the binding of gp120 to CD4. The enhanced anti-HIV activity of DNM derivatives reported here could increase the possibility of non-toxic therapeutic intervention in HIV infections.