The combined effects of anti-TNFalpha antibody and IL-1 receptor antagonist in human rheumatoid arthritis synovial membrane

TNFalpha and IL-1 are the pivotal cytokines involved in rheumatoid arthritis (RA). More recently, the biological therapy targeting TNFalpha or IL-1 has been impressively effective for many RA patients, however, it remains insufficient in some patients. In the present study, we examined the combined effects of two agents against TNFalpha and IL-1 in human RA synovial membrane. Synovial explants (an ex vivo model) and synovial fibroblasts (an in vitro model) were prepared from 11 RA patients, and then anti-TNFalpha antibody (Anti-TNFalpha) and IL-1 receptor antagonist (IL-1Ra), either alone or in combination, were added to the synovial explants and fibroblasts. IL-6 and MMP-3 production were measured after incubation. As a result, their production significantly decreased by the combination of agents compared with the control group in both the synovial explants and fibroblasts. The efficacy of this combination was also observed for IL-6 production compared with each agent alone in the synovial explants, and for IL-6 and MMP-3 production compared with each agent alone in the synovial fibroblasts. Therefore, the combination of Anti-TNFalpha and IL-1Ra appears more beneficial in synovial membrane, particularly when compared with a single agent alone.     

A detailed unfolding pathway of a (beta/alpha)8-barrel protein as studied by molecular dynamics simulations

The (beta/alpha)(8)-barrel is the most common protein fold. Similar structural properties for folding intermediates of (beta/alpha)(8)-barrel proteins involved in tryptophan biosynthesis have been reported in a number of experimental studies; these intermediates have the last two beta-strands and three alpha-helices partially unfolded, with other regions of the polypeptide chain native-like in conformation. To investigate the detailed folding/unfolding pathways of these (beta/alpha)(8)-barrel proteins, temperature-induced unfolding simulations of N-(5'-phosphoribosyl)anthranilate isomerase from Escherichia coli were carried out using a special-purpose parallel computer system. Unfolding simulations at five different temperatures showed a sequential unfolding pathway comprised of several events. Early events in unfolding involved disruption of the last two strands and three helices, producing an intermediate ensemble similar to those detected in experimental studies. Then, denaturation of the first two betaalpha units and separation of the sixth strand from the fifth took place independently. The remaining central betaalphabetaalphabeta module persisted the longest during all simulations, suggesting an important role for this module as the incipient folding scaffold. Our simulations also predicted the presence of a nucleation site, onto which several hydrophobic residues condensed forming the foundation for the central betaalphabetaalphabeta module.     

Selective inhibition of acetylcholinesterase in the cerebellum and hippocampus of mice following an acute treatment with malathion

Adult male ICR mice were treated by intraperitoneal injection with 250 mg/kg of bodyweight of commercial malathion (a dose corresponding to 1/12 the LD50). After 6 h, acetylcholinesterase (AChE) activity in blood, liver, and six brain regions was determined. A statistically significant inhibition was observed in whole blood (23%), liver (21%), and, in particular, the central nervous system; the greatest degree of AChE inhibition was observed in the cerebellum (45%), followed by the hippocampus (29%). There was no significant change in AChE activity in the caudate putamen, frontal cortex, midbrain, or pons medulla. These results demonstrate that the magnitude of AChE inhibition in peripheral tissues does not accurately reflect the central-inhibitory effects of malathion on AChE activity in specific brain regions.     

Envelope Interactions in Multi-Channel Amplitude Modulation Frequency Discrimination by Cochlear Implant Users

Rationale

Previous cochlear implant (CI) studies have shown that single-channel amplitude modulation frequency discrimination (AMFD) can be improved when coherent modulation is delivered to additional channels. It is unclear whether the multi-channel advantage is due to increased loudness, multiple envelope representations, or to component channels with better temporal processing. Measuring envelope interference may shed light on how modulated channels can be combined.

Methods

In this study, multi-channel AMFD was measured in CI subjects using a 3-alternative forced-choice, non-adaptive procedure (“which interval is different?”). For the reference stimulus, the reference AM (100 Hz) was delivered to all 3 channels. For the probe stimulus, the target AM (101, 102, 104, 108, 116, 132, 164, 228, or 256 Hz) was delivered to 1 of 3 channels, and the reference AM (100 Hz) delivered to the other 2 channels. The spacing between electrodes was varied to be wide or narrow to test different degrees of channel interaction.

Results

Results showed that CI subjects were highly sensitive to interactions between the reference and target envelopes. However, performance was non-monotonic as a function of target AM frequency. For the wide spacing, there was significantly less envelope interaction when the target AM was delivered to the basal channel. For the narrow spacing, there was no effect of target AM channel. The present data were also compared to a related previous study in which the target AM was delivered to a single channel or to all 3 channels. AMFD was much better with multiple than with single channels whether the target AM was delivered to 1 of 3 or to all 3 channels. For very small differences between the reference and target AM frequencies (2–4 Hz), there was often greater sensitivity when the target AM was delivered to 1 of 3 channels versus all 3 channels, especially for narrowly spaced electrodes.

Conclusions

Besides the increased loudness, the present results also suggest that multiple envelope representations may contribute to the multi-channel advantage observed in previous AMFD studies. The different patterns of results for the wide and narrow spacing suggest a peripheral contribution to multi-channel temporal processing. Because the effect of target AM frequency was non-monotonic in this study, adaptive procedures may not be suitable to measure AMFD thresholds with interfering envelopes. Envelope interactions among multiple channels may be quite complex, depending on the envelope information presented to each channel and the relative independence of the stimulated channels.     

Cutting edge: Calcium/Calmodulin-dependent protein kinase type IV is essential for mesangial cell proliferation and lupus nephritis

Renal involvement in systemic lupus erythematosus remains a major cause of morbidity and mortality. Although immune parameters that instigate renal damage have been characterized, their link to local processes, which execute tissue damage, is poorly understood. Using genetic-deletion and pharmacological-inhibition approaches, we demonstrated that calcium/calmodulin-dependent protein kinase type IV, which contributes to altered cytokine production in systemic lupus erythematosus patients, controls spontaneous and platelet-derived growth factor-stimulated mesangial cell proliferation and promotes IL-6 production through AP-1. Our studies identified calcium/calmodulin-dependent protein kinase type IV as a valuable treatment target for lupus nephritis and point out the importance of local kidney factors in the expression of tissue damage that, if properly targeted, should enhance clinical benefit and limit toxicity.     

Elucidation of the c-Jun N-terminal kinase pathway mediated by Estein-Barr virus-encoded latent membrane protein 1

Epstein-Barr virus (EBV) is associated with several human diseases including infectious mononucleosis and nasopharyngeal carcinoma. EBV-encoded latent membrane protein 1 (LMP1) is oncogenic and indispensable for cellular transformation caused by EBV. Expression of LMP1 in host cells constitutively activates both the c-Jun N-terminal kinase (JNK) and NF-kappaB pathways, which contributes to the oncogenic effect of LMP1. However, the underlying signaling mechanisms are not very well understood. Based mainly on overexpression studies with various dominant-negative constructs, LMP1 was generally thought to functionally mimic members of the tumor necrosis factor (TNF) receptor superfamily in signaling. In contrast to the prevailing paradigm, using embryonic fibroblasts from different knockout mice and the small interfering RNA technique, we find that the LMP1-mediated JNK pathway is distinct from those mediated by either TNF-alpha or interleukin-1. Moreover, we have further elucidated the LMP1-mediated JNK pathway by demonstrating that LMP1 selectively utilizes TNF receptor-associated factor 6, TAK1/TAB1, and c-Jun N-terminal kinase kinases 1 and 2 to activate JNK.     

Time Courses of Changes in Phospho- and Total- MAP Kinases in the Cochlea after Intense Noise Exposure

Mitogen-activated protein kinases (MAP kinases) are intracellular signaling kinases activated by phosphorylation in response to a variety of extracellular stimuli. Mammalian MAP kinase pathways are composed of three major pathways: MEK1 (mitogen-activated protein kinase kinase 1)/ERK 1/2 (extracellular signal-regulated kinases 1/2)/p90 RSK (p90 ribosomal S6 kinase), JNK (c-Jun amino (N)-terminal kinase)/c-Jun, and p38 MAPK pathways. These pathways coordinately mediate physiological processes such as cell survival, protein synthesis, cell proliferation, growth, migration, and apoptosis. The involvement of MAP kinase in noise-induced hearing loss (NIHL) has been implicated in the cochlea; however, it is unknown how expression levels of MAP kinase change after the onset of NIHL and whether they are regulated by transient phosphorylation or protein synthesis. CBA/J mice were exposed to 120-dB octave band noise for 2 h. Auditory brainstem response confirmed a component of temporary threshold shift within 0–24 h and significant permanent threshold shift at 14 days after noise exposure. Levels and localizations of phospho- and total- MEK1/ERK1/2/p90 RSK, JNK/c-Jun, and p38 MAPK were comprehensively analyzed by the Bio-Plex® Suspension Array System and immunohistochemistry at 0, 3, 6, 12, 24 and 48 h after noise exposure. The phospho-MEK1/ERK1/2/p90 RSK signaling pathway was activated in the spiral ligament and the sensory and supporting cells of the organ of Corti, with peaks at 3–6 h and independently of regulations of total-MEK1/ERK1/2/p90 RSK. The expression of phospho-JNK and p38 MAPK showed late upregulation in spiral neurons at 48 h, in addition to early upregulations with peaks at 3 h after noise trauma. Phospho-p38 MAPK activation was dependent on upregulation of total-p38 MAPK. At present, comprehensive data on MAP kinase expression provide significant insight into understanding the molecular mechanism of NIHL, and for developing therapeutic models for acute sensorineural hearing loss.     

Osteogenic Differentiation Capacity of Human Skeletal Muscle-Derived Progenitor Cells

Heterotopic ossification (HO) is defined as the formation of ectopic bone in soft tissue outside the skeletal tissue. HO is thought to result from aberrant differentiation of osteogenic progenitors within skeletal muscle. However, the precise origin of HO is still unclear. Skeletal muscle contains two kinds of progenitor cells, myogenic progenitors and mesenchymal progenitors. Myogenic and mesenchymal progenitors in human skeletal muscle can be identified as CD56⁺ and PDGFRα⁺ cells, respectively. The purpose of this study was to investigate the osteogenic differentiation potential of human skeletal muscle-derived progenitors. Both CD56⁺ cells and PDGFRα⁺ cells showed comparable osteogenic differentiation potential in vitro. However, in an in vivo ectopic bone formation model, PDGFRα⁺ cells formed bone-like tissue and showed successful engraftment, while CD56⁺ cells did not form bone-like tissue and did not adapt to an osteogenic environment. Immunohistological analysis of human HO sample revealed that many PDGFRα⁺ cells were localized in proximity to ectopic bone formed in skeletal muscle. MicroRNAs (miRNAs) are known to regulate many biological processes including osteogenic differentiation. We investigated the participation of miRNAs in the osteogenic differentiation of PDGFRα⁺ cells by using microarray. We identified miRNAs that had not been known to be involved in osteogenesis but showed dramatic changes during osteogenic differentiation of PDGFRα⁺ cells. Upregulation of miR-146b-5p and -424 and downregulation of miR-7 during osteogenic differentiation of PDGFRα⁺ cells were confirmed by quantitative real-time RT-PCR. Inhibition of upregulated miRNAs, miR-146b-5p and -424, resulted in the suppression of osteocyte maturation, suggesting that these two miRNAs have the positive role in the osteogenesis of PDGFRα⁺ cells. Our results suggest that PDGFRα⁺ cells may be the major source of HO and that the newly identified miRNAs may regulate osteogenic differentiation process of PDGFRα⁺ cells.     

Enhanced antitumor activities of (-)-epigallocatechin-3-O-gallate fatty acid monoester derivatives in vitro and in vivo

(−)-Epigallocatechin-3-O-gallate (EGCG) monoesters modified with butanoyl (EGCG-C4), octanoyl (EGCG-C8), palmitoyl groups (EGCG-C16) were synthesized by a lipase-catalyzed transesterification method and their antitumor activities were investigated in vitro and in vivo. The in vitro antitumor activities of EGCG-monoester derivatives increased in an alkyl chain length-dependent manner. The cytotoxicity of EGCG, EGCG-C4, EGCG-C8 was mainly caused by H₂O₂ which was generated with their oxidation. On the other hand, EGCG-C16 was more stable than EGCG and it did not generate H₂O₂ in the cell culture medium. Furthermore, EGCG-C16 inhibited cell proliferation and induced apoptosis in the presence of catalase. EGCG-C16 was found to inhibit the phosphorylation of the epidermal growth factor receptor (EGFR), which is related to various types of tumor growth. EGCG-C16 suppressed tumor growth in vivo in colorectal tumor bearing mice in comparison to an untreated control, vector control (DMSO) and EGCG.