Biodentine Reduces Tumor Necrosis Factor Alpha-induced TRPA1 Expression in Odontoblastlike Cells

International audienceIntroduction: The transient receptor potential (TRP) ion channels have emerged as important cellular sensors in both neuronal and non-neuronal cells, with TRPA1 playing a central role in nociception and neurogenic inflammation. The functionality of TRP channels has been shown to be modulated by inflammatory cytokines. The aim of this study was to investigate the effect of inflammation on odontoblast TRPA1 expression and to determine the effect of Biodentine (Septodent, Paris, France) on inflammatory-induced TRPA1 expression. Methods: Immunohistochemistry was used to study TRPA1 expression in pulp tissue from healthy and carious human teeth. Pulp cells were differentiated to odontoblastlike cells in the presence of 2 mmol/L beta-glycerophosphate, and these cells were used in quantitative polymerase chain reaction, Western blotting, calcium imaging, and patch clamp studies. Results: Immunofluorescent staining revealed. TRPA1 expression in odontoblast cell bodies and odontoblast processes, which was more intense in carious versus healthy teeth. TRPA1 gene expression was induced in cultured odontoblastlike cells by tumor necrosis factor alpha, and this expression was significantly reduced in the presence of Biodentine. The functionality of the TRPA1 channel was shown by calcium microfluorimetry and patch clamp recording, and our results showed a significant reduction in tumor necrosis factor alpha induced TRPA1 responses after Biodentine treatment. Conclusions: In conclusion, this study showed TRPA1 to be modulated by caries-induced inflammation and that Biodentine reduced TRPA1 expression and functional responses

Physiology and Pharmacology Ca V 3.1 T-Type Ca 2þ Channels Contribute to Myogenic Signaling in Rat Retinal Arterioles

PURPOSE. Although L-type Ca 2þ channels are known to play a key role in the myogenic reactivity of retinal arterial vessels, the involvement of other types of voltage-gated Ca 2þ channels in this process remains unknown. In the present study we have investigated the contribution of T-type Ca 2þ channels to myogenic signaling in arterioles of the rat retinal microcirculation. METHODS. Confocal immunolabeling of whole-mount preparations was used to investigate the localization of Ca V 3.1-3 channels in retinal arteriolar smooth muscle cells. T-type currents and the contribution of T-type channels to myogenic signaling were assessed by whole-cell patchclamp recording and pressure myography of isolated retinal arteriole segments. RESULTS. Strong immunolabeling for Ca V 3.1 was observed on the plasma membrane of retinal arteriolar smooth muscle cells. In contrast, no expression of Ca V 3.2 or Ca V 3.3 could be detected in retinal arterioles, although these channels were present on glial cell end-feet surrounding the vessels and retinal ganglion cells, respectively. TTA-A2-sensitive T-type currents were recorded in retinal arteriolar myocytes with biophysical properties distinct from those of the L-type currents present in these cells. Inhibition of T-type channels using TTA-A2 or ML-218 dilated isolated, myogenically active, retinal arterioles. CONCLUSIONS. Ca V 3.1 T-type Ca 2þ channels are functionally expressed on arteriolar smooth muscle cells of retinal arterioles and play an important role in myogenic signaling in these vessels. The work has important implications concerning our understanding of the mechanisms controlling blood flow autoregulation in the retina and its disruption during ocular disease

Exploitation of the bilosome platform technology to formulate antibiotics and enhance efficacy of melioidosis treatments

Burkholderia pseudomallei is a Gram-negative intracellular bacterium which is recalcitrant to antibiotic therapy. There also is currently no licensed vaccine for this potentially fatal pathogen, further highlighting the requirement for better therapeutics to treat the disease melioidosis. Here we use an oral delivery platform, the bilosome to entrap already- licensed antibiotics. Bilosome-entrapped antibiotics were used to treat mice infected via the aerosol route with B. pseudomallei. When treatment was started by the oral route at 6 h post-infection and continued for 7 days, bilosome levofloxacin and bilosome doxycycline formulations were significantly more efficacious than free antibiotics in terms of survival rates. Additionally, bilosome formulated levofloxacin protected mice from antibiotic and infection induced weight loss following B. pseudomallei infection. The microbiomes of mice treated with levofloxacin were depleted of all phyla with the exception of Firmicutes, but doxycycline treatment had minimal effect on the microbiome. Encapsulation of either drug in bilosomes had no deleterious or clear advantageous effect on microbiome. This indicates that the ability of bilosomes to ameliorate antibiotic induced weight loss is not due to microbiome effects. The bilosome platform not only has potential to reduce adverse effects of orally delivered antimicrobials, but has potential for other therapeutics which may cause detrimental side-effects or require enhanced delivery

Expression of Constitutively Active Raf-1 in the Mitochondria Restores Antiapoptotic and Leukemogenic Potential of a Transformation-deficient BCR/ABL Mutant

The oncogenic BCR/ABL protein protects hematopoietic cells from apoptosis induced by growth factor deprivation, but the mechanisms are only partially understood. A BCR/ABL mutant lacking amino acids 176–426 in the BCR domain (p185ΔBCR) failed to protect interleukin 3–deprived 32Dcl3 myeloid precursor cells from apoptosis, although it possessed tyrosine kinase activity and was capable of activating the Ras-Raf-MAP kinase pathway. Compared to p185 wild-type transfectants, p185ΔBCR-transfected cells showed markedly reduced levels of Bcl-2 and expressed the hypophosphorylated, proapoptotic form of BAD. Bcl-2 expression in the mitochondrial fraction of p185ΔBCR cells was also markedly diminished and mitochondrial RAF was undetectable. In p185ΔBCR cells transfected with a mitochondria-targeted, constitutively active RAF (M-Raf) BAD was expressed in the hyperphosphorylated form and released from the mitochondria into the cytosol. p185ΔBCR/M-Raf–transfected cells were completely resistant to apoptosis induced by growth factor deprivation in vitro. Moreover, constitutive expression of dominant-negative M-Raf (K375W) enhanced the susceptibility of 32Dcl3 cells expressing wild-type BCR/ABL to apoptosis. In severe combined immunodeficiency (SCID) mice, p185ΔBCR/M-Raf double transfectants were leukemogenic, whereas cells expressing only p185ΔBCR showed no leukemogenic potential. Together, these data support the existence of a BCR/ABL-dependent pathway that leads to expression of an active RAF in the mitochondria and promotes antiapoptotic and leukemia-inducing effects of BCR/ABL

Hyperglycemia and Diabetes Downregulate the Functional Expression of TRPV4 Channels in Retinal Microvascular Endothelium

Retinal endothelial cell dysfunction is believed to play a key role in the etiology and pathogenesis of diabetic retinopathy. Numerous studies have shown that TRPV4 channels are critically involved in maintaining normal endothelial cell function. In the current paper, we demonstrate that TRPV4 is functionally expressed in the endothelium of the retinal microcirculation and that both channel expression and activity is downregulated by hyperglycaemia. Quantitative PCR and immunostaining demonstrated molecular expression of TRPV4 in cultured bovine retinal microvascular endothelial cells (RMECs). Functional TRPV4 activity was assessed in cultured RMECs from endothelial Ca2+-responses recorded using fura-2 microfluorimetry and electrophysiological recordings of membrane currents. The TRPV4 agonist 4α-phorbol 12,13-didecanoate (4-αPDD) increased [Ca2+]i in RMECs and this response was largely abolished using siRNA targeted against TRPV4. These Ca2+-signals were completely inhibited by removal of extracellular Ca2+, confirming their dependence on influx of extracellular Ca2+. The 4-αPDD Ca2+-response recorded in the presence of cyclopiazonic acid (CPA), which depletes the intracellular stores preventing any signal amplification through store release, was used as a measure of Ca2+-influx across the cell membrane. This response was blocked by HC067047, a TRPV4 antagonist. Under voltage clamp conditions, the TRPV4 agonist GSK1016790A stimulated a membrane current, which was again inhibited by HC067047. Following incubation with 25 mM D-glucose TRPV4 expression was reduced in comparison with RMECs cultured under control conditions, as were 4αPDD-induced Ca2+-responses in the presence of CPA and ion currents evoked by GSK1016790A. Molecular expression of TRPV4 in the retinal vascular endothelium of 3 months' streptozotocin-induced diabetic rats was also reduced in comparison with that in age-matched controls. We conclude that hyperglycaemia and diabetes reduce the molecular and functional expression of TRPV4 channels in retinal microvascular endothelial cells. These changes may contribute to diabetes induced endothelial dysfunction and retinopathy