Endogenous CD8+ T cell expansion during regression of monoclonal EBV-associated posttransplant lymphoproliferative disorder.

There are experimental data which suggest that the primary immune effector cell responsible for maintaining immune surveillance against the outgrowth of EBV-transformed B cells in humans is the CTL, but in vivo proof of this is lacking. In this study we perform a series of cellular and molecular assays to characterize an autologous, endogenous immune response against a transplantation-associated, monoclonal, EBV+ posttransplant lymphoproliferative disorder (PTLD). Following allogeneic bone marrow transplantation, a patient developed a monoclonal PTLD of donor B cell origin. With a decrease in immune suppression, we document the emergence of endogenous, donor-derived CD3+CD8+ CTLs, followed by regression of the PTLD. The TCR Vbeta repertoire went from a polyclonal pattern prior to the development of PTLD to a restricted TCR Vbeta pattern during the outgrowth and regression of PTLD. Donor-derived CD3+CD8+ T lymphocytes displayed MHC class I-restricted cytolytic activity against the autologous EBV+ B cells ex vivo without additional in vitro sensitization. The striking temporal relationship between the endogenous expansion of a TCR Vbeta-restricted, CD3+CD8+ population of MHC class I-restricted CTL, and the regression of an autologous monoclonal PTLD, provides direct evidence in humans that endogenous CD3+CD8+ CTLs can be responsible for effective immune surveillance against malignant transformation of EBV+ B cells.     

Characterisation of Ilomastat for Prolonged Ocular Drug Release

We are developing tablet dosage forms for implantation directly into the subconjunctival space of the eye. The matrix metalloproteinase inhibitor, ilomastat, has previously been shown to be efficacious at suppressing scarring following glaucoma filtration surgery (GFS). We report on the physical characterisation of ilomastat which is being developed for ocular implantation. Since ilomastat is being considered for implantation it is necessary to examine its polymorphs and their influence on aspects of the in vitro drug release profile. X-ray powder diffraction identified two polymorphs of ilomastat from different commercial batches of the compound. Tablets were prepared from the two different polymorphs. Isothermal perfusion calorimetry was used to show that amorphous content is not increased during tablet formulation. The melting points of the two polymorphs are 188 and 208°C as determined by differential scanning calorimetry. Utilising single crystal X-ray diffraction, the structural conformations and packing arrangements of the different polymorphs were determined. The orthorhombic crystal crystallised as a monohydrate while the second monoclinic crystal form is non-solvated. Ilomastat tablets prepared from the two different solid forms exhibited similar drug release profiles in vitro under conditions mimicking the aqueous composition, volume and flow of the subconjunctival space after GFS. This suggests that a reproducible dose at each time point during release after implantation should be achievable in vivo with ilomastat tablets prepared from the two polymorphs identified.     

DHFR silence alleviated the development of liver fibrosis by affecting the crosstalk between hepatic stellate cells and macrophages

Liver fibrogenesis is a dynamic cellular and tissue process which has the potential to progress into cirrhosis of even liver cancer and liver failure. The activation of hepatic stellate cells (HSCs) is the central event underlying liver fibrosis. Besides, hepatic macrophages have been proposed as potential targets in combatting fibrosis. As for the relationship between HSCs and hepatic macrophages in liver fibrosis, it is generally considered that macrophages promoted liver fibrosis via activating HSCs. However, whether activated HSCs could in turn affect macrophage polarization has rarely been studied. In this study, mRNAs with significant differences were explored using exosomal RNA-sequencing of activated Lx-2 cells and normal RNA-sequencing of DHFR loss-of-function Lx-2 cell models. Cell functional experiments in both Lx-2 cells and macrophages animal model experiments were performed. The results basically confirmed exosomes secreted from activated HSCs could promote M1 polarization of macrophages further. Exosome harbouring DHFR played an important role in this process. DHFR silence in HSCs could decrease Lx-2 activation and M1 polarization of M0 macrophages and then alleviate the development of liver fibrosis both in vitro and vivo. Our work brought a new insight that exosomal DHFR derived from HSCs had a crucial role in crosstalk between HSCs activation and macrophage polarization, which may be a potential therapeutic target in liver fibrosis.     

Serum miR-128-2 Serves as a Prognostic Marker for Patients with Hepatocellular Carcinoma

Circulating miRNAs are promising biomarkers for predicting the aggressiveness of hepatocellular carcinoma (HCC). We aimed to identify differentially expressed miRNAs in the serum of HCC patients with different Barcelona Clinic Liver Cancer (BCLC) stage, and to investigate the potential of serum miRNAs as biomarkers for patient outcomes. In the discovery stage, TaqMan Low-Density Array was used to test the difference in levels of serum miRNAs between 20 patients with portal vein tumor thrombosis (PVTT) and 20 patients without PVTT. The detected serum miRNAs then were validated in 182 patients. Fifteen serum miRNAs showed more than two-fold higher expression in patients with PVTT, and miR-128-2 was found to be significantly up-regulated and was selected for further validation. In the validation stage, patients were divided into two groups with low or high serum miR-128-2 using the median expression level of all 182 cases as the cut-off point. Kaplan-Meier analysis revealed that patients with low level of serum miR-128-2 had favorable trends of survival (log rank = 13.031, p < 0.001). The median survivals for patients with a low and high level of serum miR-128-2 were 625 (95% CI, 527–722) days and 426 (95% CI, 362–491) days, respectively. MiR-128-2 was also an independent factor of overall survival (p = 0.001, HR 2.793, 95%CI 1.550, 5.033). Serum levels of the ubiquitously expressed miR-128-2 showed no significant correlation with parameters of liver damage or liver function. In addition, expressions of miR-128-2 in HCC tissues were up-regulated in comparison with adjacent non-tumor tissues. In conclusion, serum level of miR-128-2 serves as a noninvasive biomarker for the overall survival of patients with hepatocellular carcinoma.     

Up-Regulatory Effects of Curcumin on Large Conductance Ca2+-Activated K+ Channels

Large conductance Ca²⁺-activated potassium channels (BK) are targets for research that explores therapeutic means to various diseases, owing to the roles of the channels in mediating multiple physiological processes in various cells and tissues. We investigated the pharmacological effects of curcumin, a compound isolated from the herb Curcuma longa, on BK channels. As recorded by whole-cell patch-clamp, curcumin increased BK (α) and BK (α+β1) currents in transfected HEK293 cells as well as the current density of BK in A7r5 smooth muscle cells in a dose-dependent manner. By incubating with curcumin for 24 hours, the current density of exogenous BK (α) in HEK293 cells and the endogenous BK in A7r5 cells were both enhanced notably, though the steady-state activation of the channels did not shift significantly, except for BK (α+β1). Curcumin up-regulated the BK protein expression without changing its mRNA level in A7r5 cells. The surface expression and the half-life of BK channels were also increased by curcumin in HEK293 cells. These effects of curcumin were abolished by MG-132, a proteasome inhibitor. Curcumin also increased ERK 1/2 phosphorylation, while inhibiting ERK by U0126 attenuated the curcumin-induced up-regulation of BK protein expression. We also observed that the curcumin-induced relaxation in the isolated rat aortic rings was significantly attenuated by paxilline, a BK channel specific blocker. These results show that curcumin enhances the activity of the BK channels by interacting with BK directly as well as enhancing BK protein expression through inhibiting proteasomal degradation and activating ERK signaling pathway. The findings suggest that curcumin is a potential BK channel activator and provide novel insight into its complicated pharmacological effects and the underlying mechanisms.     

Long noncoding RNA PTENP1 affects the recovery of spinal cord injury by regulating the expression of miR-19b and miR-21

Exosomes derived from differentiated P12 cells and MSCs were proved to suppress apoptosis of neuron cells, and phosphatase and tensin homolog pseudogene 1 (PTENP1) was reported to inhibit cell proliferation. In this study, we aimed to investigate the role of PTENP1 in the process of post-spinal cord injury (SCI) recovery, so as to evaluate the therapeutic effects of exosomes derived from MSCs transfected with PTENP1 short hairpin RNA (shRNA), as a type of novel biomarkers in the treatment of SCI. Electron microscopy was used to observe the morphology of different exosomes. Real-time polymerase chain reaction and western blot, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, flow cytometry, Nissl staining, immunohistochemistry assay, and terminal deoxynucleotidyl transferase dUTP nick end labeling assay were conducted to investigate and validate the underlying molecular signaling pathway. PTENP1-shRNA downregulated PTENP1 and PTEN while upregulating miR-21 and miR-19b. PTENP1-shRNA also accelerated cell apoptosis and reduced cell viability. In addition, PTENP1 reduced the miR-21 and miR-19b expression by directly targeting miR-21 and miR-19b. Meanwhile, both miR-21 and miR-19b reduced the expression of PTEN by directly targeting the 3′-untranslated region of PTEN. Furthermore, PTEN level and apoptosis index of neuron cells was the highest in the SCI group, while the treatment with exosomes+PTENP1-shRNA reduced the PTEN expression to a level similar to that in the sham group. Finally, PTENP1 inhibited miR-21 and miR-19b expression but upregulated PTEN expression. The upregulation of miR-21/miR-19b also suppressed the apoptosis of neuron cells by downregulating the PTEN expression. PTENP1 is involved in the recovery of SCI by regulating the expression of miR-19b and miR-21, and exosomes from PTENP1-shRNA-transfected cells may be used as a novel biomarker in SCI treatment.     

Integrative Approach to Analyze Biodiversity and Anti-Inflammatory Bioactivity of Wedelia Medicinal Plants

For the development of “medical foods” and/or botanical drugs as defined USA FDA, clear and systemic characterizations of the taxonomy, index phytochemical components, and the functional or medicinal bioactivities of the reputed or candidate medicinal plant are needed. In this study, we used an integrative approach, including macroscopic and microscopic examination, marker gene analysis, and chemical fingerprinting, to authenticate and validate various species/varieties of Wedelia, a reputed medicinal plant that grows naturally and commonly used in Asian countries. The anti-inflammatory bioactivities of Wedelia extracts were then evaluated in a DSS-induced murine colitis model. Different species/varieties of Wedelia exhibited distinguishable morphology and histological structures. Analysis of the ribosomal DNA internal transcribed spacer (ITS) region revealed significant differences among these plants. Chemical profiling of test Wedelia species demonstrated candidate index compounds and distinguishable secondary metabolites, such as caffeic acid derivatives, which may serve as phytochemical markers or index for quality control and identification of specific Wedelia species. In assessing their effect on treating DSS induced-murine colitis, we observed that only the phytoextract from W. chinensis species exhibited significant anti-inflammatory bioactivity on DSS-induced murine colitis among the various Wedelia species commonly found in Taiwan. Our results provide a translational research approach that may serve as a useful reference platform for biotechnological applications of traditional phytomedicines. Our findings indicate that specific Wedelia species warrant further investigation for potential treatment of human inflammatory bowel disease.     

The oncolytic peptide LTX-315 triggers necrotic cell death

The oncolytic peptide LTX-315 has been designed for killing human cancer cells and turned out to stimulate anti-cancer immune responses when locally injected into tumors established in immunocompetent mice. Here, we investigated the question whether LTX-315 induces apoptosis or necrosis. Transmission electron microscopy or morphometric analysis of chromatin-stained tumor cells revealed that LTX-315 failed to induce apoptotic nuclear condensation and rather induced a necrotic phenotype. Accordingly, LTX-315 failed to stimulate the activation of caspase-3, and inhibition of caspases by means of Z-VAD-fmk was unable to reduce cell killing by LTX-315. In addition, 2 prominent inhibitors of regulated necrosis (necroptosis), namely, necrostatin-1 and cycosporin A, failed to reduce LTX-315-induced cell death. In conclusion, it appears that LTX-315 triggers unregulated necrosis, which may contribute to its pro-inflammatory and pro-immune effects.