Stimulation of Inositol Phosphate Production by Neurotensin in Neuroblastoma N1E115 Cells: Implication of GTP-Binding Proteins and Relationship with the Cyclic GMP Response

The association of neurotensin to its receptor in differentiated neuroblastoma N1E115 cells led to a fast and transitory increase of the intracellular concentration in inositol triphosphate and inositol biphosphate, followed by a slower and more stable increase inositol monophosphate. The action of inositol 1,4,5-triphosphate on digitonin-permeabilized N1E115 cells resulted in a stimulation of cyclic GMP levels that mimicked that induced by neurotensin. Therefore, the cyclic GMP stimulation is probably a consequence of the initial inositol triphosphate formation triggered by neurotensin. Fluoroaluminate ions and pertussis toxin had the capacity to modulate positively and negatively, respectively, the formation of inositol triphosphate induced by neurotensin, indicating that GTP-binding proteins are involved in the regulation of inositol phosphate levels by neurotensin receptors.     

The Ras antagonist, farnesylthiosalicylic acid (FTS), decreases fibrosis and improves muscle strength in dy/dy mouse model of muscular dystrophy

The Ras superfamily of guanosine-triphosphate (GTP)-binding proteins regulates a diverse spectrum of intracellular processes involved in inflammation and fibrosis. Farnesythiosalicylic acid (FTS) is a unique and potent Ras inhibitor which decreased inflammation and fibrosis in experimentally induced liver cirrhosis and ameliorated inflammatory processes in systemic lupus erythematosus, neuritis and nephritis animal models. FTS effect on Ras expression and activity, muscle strength and fibrosis was evaluated in the dy(2J)/dy(2J) mouse model of merosin deficient congenital muscular dystrophy. The dy(2J)/dy(2J) mice had significantly increased RAS expression and activity compared with the wild type mice. FTS treatment significantly decreased RAS expression and activity. In addition, phosphorylation of ERK, a Ras downstream protein, was significantly decreased following FTS treatment in the dy(2J)/dy(2J) mice. Clinically, FTS treated mice showed significant improvement in hind limb muscle strength measured by electronic grip strength meter. Significant reduction of fibrosis was demonstrated in the treated group by quantitative Sirius Red staining and lower muscle collagen content. FTS effect was associated with significantly inhibition of both MMP-2 and MMP-9 activities. We conclude that active RAS inhibition by FTS was associated with attenuated fibrosis and improved muscle strength in the dy(2J)/dy(2J) mouse model of congenital muscular dystrophy.     

Impaired Quality of the Hepatitis B Virus (HBV)-Specific T-Cell Response in Human Immunodeficiency Virus Type 1-HBV Coinfection

Hepatits B virus (HBV)-specific T cells play a key role both in the control of HBV replication and in the pathogenesis of liver disease. Human immunodeficiency virus type 1 (HIV-1) coinfection and the presence or absence of HBV e (precore) antigen (HBeAg) significantly alter the natural history of chronic HBV infection. We examined the HBV-specific T-cell responses in treatment-naïve HBeAg-positive and HBeAg-negative HIV-1-HBV-coinfected (n = 24) and HBV-monoinfected (n = 39) Asian patients. Peripheral blood was stimulated with an overlapping peptide library for the whole HBV genome, and tumor necrosis factor alpha and gamma interferon cytokine expression in CD8⁺ T cells was measured by intracellular cytokine staining and flow cytometry. There was no difference in the overall magnitude of the HBV-specific T-cell responses, but the quality of the response was significantly impaired in HIV-1-HBV-coinfected patients compared with monoinfected patients. In coinfected patients, HBV-specific T cells rarely produced more than one cytokine and responded to fewer HBV proteins than in monoinfected patients. Overall, the frequency and quality of the HBV-specific T-cell responses increased with a higher CD4⁺ T-cell count (P = 0.018 and 0.032, respectively). There was no relationship between circulating HBV-specific T cells and liver damage as measured by activity and fibrosis scores, and the HBV-specific T-cell responses were not significantly different in patients with either HBeAg-positive or HBeAg-negative disease. The quality of the HBV-specific T-cell response is impaired in the setting of HIV-1-HBV coinfection and is related to the CD4⁺ T-cell count.There are 40 million people worldwide infected with human immunodeficiency virus type 1 (HIV-1), and 6 to 15% of HIV-1-infected patients are also chronically infected with hepatitis B virus (HBV) (13, 20, 35, 38, 40-42, 47, 50, 61, 69). The highest rates of coinfection with HIV-1 and HBV are in Asia and Africa, where HBV is endemic (33, 68). Following the introduction of highly active antiretroviral therapy (HAART), liver disease is now the major cause of non-AIDS-related deaths in HIV-1-infected patients (12, 13, 38, 59, 65).Coinfection of HBV with HIV-1 alters the natural history of HBV infection. Individuals with HIV-1-HBV coinfection seroconvert from HBV e (precore) antigen (HBeAg) to HBV e antibody less frequently and have higher HBV DNA levels but lower levels of alanine aminotransferase (ALT) and milder necroinflammatory activity on histology than those infected with HBV alone (18, 26, 49). Progression to cirrhosis, however, seems to be more rapid and more common, and liver-related mortality is higher, in HIV-1-HBV coinfection than with either infection alone (47, 59). HBeAg is an accessory protein of HBV and is not required for viral replication or infection; however, chronic HBV infection typically is divided into two distinct phases: HBeAg positive and HBeAg negative (reviewed in reference 15). Most natural history studies of HIV-1-HBV coinfection to date have primarily focused on HBeAg-positive patients from non-Asian countries (23, 44, 46).We previously developed an overlapping peptide library for the HBV genome to detect HBV-specific CD4⁺ and CD8⁺ T-cell responses to all HBV gene products from multiple HBV genotypes (17). In a small cross-sectional study of patients recruited in Australia, we found that in coinfected patients, HBV-specific CD4⁺ T-cell responses, as measured by gamma interferon (IFN-γ) production, were diminished compared to those seen in HBV-monoinfected patients (17). However, patients had varying lengths of exposure to anti-HBV-active HAART at the time of analysis. In this study, therefore, we aimed to characterize the HBV-specific T-cell response in untreated HBeAg-positive and HBeAg-negative HIV-1-HBV-coinfected patients and to determine the relationship between the HBV-specific immune response, HBeAg status, and liver disease.     

Split immunity: immune inhibition of rat gliomas by subcutaneous exposure to unmodified live tumor cells

Gliomas that grow uninhibited in the brain almost never metastasize outside the CNS. The rare occurrences of extracranial metastasis are usually associated with a suppressed immune system. This observation raises the possibility that some gliomas might not grow outside the CNS due to an inherent immune response, We report in this study that the highly malignant F98 Fischer rat undifferentiated glioma, which grows aggressively in the brain, spontaneously regresses when injected live s.c. We found that this regression is immune-mediated and that it markedly enhances the survival or cures rats challenged with the same tumor intracranially either before or after the s.c. live-cell treatment. Adoptive transfer experiments showed the effect was immune-mediated and that the CD8 T cell fraction, which exhibited direct tumor cytotoxicity, was more effective than the CD4 T cell fraction in mediating resistance to intracranial challenge of naive rats. Brain tumors from treated rats exhibited enhanced CD3(+)CD8(+)CD4(-) and CD3(+)CD4(+)CD8(-) T cell infiltration and IFN-γ secretion. The results in the F98 glioma were corroborated in the Lewis rat CNS-1 astrocytoma. In both tumor models, s.c. treatment with live cells was significantly better than immunization with irradiated cells. We propose in this study a location-based immunotherapeutic phenomenon we term "split immunity": a tumor that thrives in an immune-privileged site may be inhibited by injecting live, unmodified tumor cells into a site that is not privileged, generating protective immunity that spreads back to the privileged site. Split immunity could explain several long-standing paradoxes regarding the lack of overt extracranial metastasis in patients with primary brain tumors.     

Grapevine petioles are more sensitive to drought induced embolism than stems: evidence from in vivo MRI and microcomputed tomography observations of hydraulic vulnerability segmentation

The ‘hydraulic vulnerability segmentation’ hypothesis predicts that expendable distal organs are more susceptible to water stress‐induced embolism than the main stem of the plant. In the current work, we present the first in vivo visualization of this phenomenon. In two separate experiments, using magnetic resonance imaging or synchrotron‐based microcomputed tomography, grapevines (Vitis vinifera) were dehydrated while simultaneously scanning the main stems and petioles for the occurrence of emboli at different xylem pressures (Ψ_x). Magnetic resonance imaging revealed that 50% of the conductive xylem area of the petioles was embolized at a Ψ_x of ?1.54 MPa, whereas the stems did not reach similar losses until ?1.9 MPa. Microcomputed tomography confirmed these findings, showing that approximately half the vessels in the petioles were embolized at a Ψ_x of ?1.6 MPa, whereas only few were embolized in the stems. Petioles were shown to be more resistant to water stress‐induced embolism than previously measured with invasive hydraulic methods. The results provide the first direct evidence for the hydraulic vulnerability segmentation hypothesis and highlight its importance in grapevine responses to severe water stress. Additionally, these data suggest that air entry through the petiole into the stem is unlikely in grapevines during drought.     

Amplified Inhibition of Stellate Cell Activation Pathways by PPAR-γ, RAR and RXR Agonists

Peroxisome proliferator activator receptors (PPAR) ligands such as 15-Δ12,13-prostaglandin L(2) [PJ] and all trans retinoic acid (ATRA) have been shown to inhibit the development of liver fibrosis. The role of ligands of retinoic X receptor (RXR) and its ligand, 9-cis, is less clear. The purpose of this study was to investigate the effects of combined treatment of the three ligends, PJ, ATRA and 9-cis, on key events during liver fibrosis in rat primary hepatic stellate cells (HSCs). We found that the anti-proliferative effect of the combined treatment of PJ, ATRA and 9-cis on HSCs was additive. Further experiments revealed that this inhibition was due to cell cycle arrest at the G₀/G₁ phase as demonstrated by FACS analysis. In addition, the combined treatment reduced cyclin D1 expression and increased p21 and p27 protein levels. Furthermore, we found that the three ligands down regulated the phosphorylation of mTOR and p70^S6K. The activation of HSCs was also inhibited by the three ligands as shown by inhibition of vitamin A lipid droplets depletion from HSCs. Studies using real time PCR and western blot analysis showed marked inhibition of collagen Iα1 and αSMA by the combination of the three ligands. These findings suggest that the combined use of PJ, ATRA and 9-cis causes inhibition of cell proliferation by cell cycle arrest and down-regulation of fibrotic markers to a greater extent compared to each of the ligands alone.     

Structural variability in manganese(II) complexes of N,N′-bis(2-pyridinylmethylene) ethane (and propane) diamine ligands

Manganese(II) complexes, Mn₂L¹₃(ClO₄)₄, MnL¹(H₂O)₂(ClO₄)₂, MnL²(H₂O)₂(ClO₄)₂, and {(μ-Cl)MnL²(PF₆)}₂ based on N,N′-bis(2-pyridinylmethylene) ethanediamine (L¹) and N,N′-bis(2-pyridinylmethylene) propanediamine (L²) ligands have been prepared and characterized. The single crystal X-ray diffraction analysis of Mn₂L²₃(ClO₄)₄ shows that each of the two Mn(II) ion centers with a Mn-Mn distance of 7.15 Å are coordinated by one ligand while a common third ligand bridges the metal centers. Solid-state magnetic susceptibility measurements as well as DFT calculations confirm that each of the manganese centers is high-spin S = 5/2. The electronic structure obtained shows no orbital overlap between the Mn(II) centers indicating that the observed weak antiferromagentism is a result of through space interactions between the two Mn(II) centers. Under different reaction conditions, L¹ and Mn(II) yielded a one-dimensional polymer, MnL¹(H₂O)₂(ClO₄)₂. Ligand L² when reacted with manganese(II) perchlorate gives contrarily to L¹ mononuclear MnL²(H₂O)₂(ClO₄)₂ complex. The analysis of the structural properties of the MnL²(H₂O)₂(ClO₄)₂ lead to the design of dinuclear complex {(μ-Cl)MnL²(PF₆)} where two chlorine atoms were utilized as bridging moieties. This complex has a rhomboidal Mn₂Cl₂ core with a Mn-Mn distance of 3.726 Å. At room temperature {(μ-Cl)MnL²(PF₆)} is ferromagnetic with observed μ_eff = 4.04 μ_B per Mn(II) ion. With cooling, μ_eff grows reaching 4.81 μ_B per Mn(II) ion at 8 K, and then undergoes ferromagnetic-to-antiferromagnetic phase transition.