Depletion of CD4+ CD25+ regulatory T cells promotes CCL21-mediated antitumor immunity.

CCL21 is known to attract dendritic cells (DCs) and T cells that may reverse tumor-mediated immune suppression. The massive infiltration of tumors by regulatory T cells (Tregs) prevents the development of a successful helper immune response. In this study, we investigated whether elimination of CD4(+) CD25(+) Tregs in the tumor microenvironment using anti-CD25 monoclonal antibodies (mAbs) was capable of enhancing CCL21-mediated antitumor immunity in a mouse hepatocellular carcinoma (HCC) model. We found that CCL21 in combination with anti-CD25 mAbs (PC61) resulted in improved antitumor efficacy and prolonged survival, not only inhibited tumor angiogenesis and cell proliferation, but also led to significant increases in the frequency of CD4(+), CD8(+) T cells and CD11c(+) DCs within the tumor, coincident with marked induction of tumor-specific CD8(+) cytotoxic T lymphocytes (CTLs) at the local tumor site. The intratumoral immune responses were accompanied by the enhanced elaboration of IL-12 and IFN-γ, but reduced release of the immunosuppressive mediators IL-10 and TGF-ß1. The results indicated that depletion of Tregs in the tumor microenvironment could enhance CCL21-mediated antitumor immunity, and CCL21 combined with anti-CD25 mAbs may be a more effective immunotherapy to promote tumor rejection.

Erythropoietin inhibits gamma-irradiation-induced apoptosis by upregulation of Bcl-2 and decreasing the activation of caspase 3 in human UT-7/erythropoietin cell line.

1. Erythropoietin (EPO) can reverse radiotherapy-induced anaemia by stimulating bone marrow cells to produce erythrocytes. However, there are limited studies that address the mechanisms by which EPO exerts its beneficial effects in radiotherapy-induced anaemia. In the present study, we used a human bone marrow-derived EPO-dependent leukaemia cell line UT-7/EPO that progressed further in erythroid development to evaluate the anti-apoptotic effects of EPO on irradiated human erythroid progenitor. 2. The UT-7/EPO cells exposed to gamma-irradiation were cultured in the presence or absence of EPO at a concentration of 7 U/mL. The cell viability, cell apoptosis and the expression of apoptosis-related proteins Bcl-2, Bax and caspase 3 were examined. 3. The results showed that EPO protected the viability of human UT-7/EPO cells exposed to gamma-irradiation. EPO significantly inhibited gamma-irradiation-induced apoptosis in human UT-7/EPO cells: a significant decrease in the percentage of apoptotic cells was observed (62, 69 and 62% at 24, 48 and 72 h, respectively). Furthermore, EPO significantly increased the expression of Bcl-2 protein and the relative Bcl-2/Bax ratio, and decreased the activation of caspase 3 and formation of the p17 and p12 cleavage in similar conditions. 4. In conclusion, EPO exerts anti-apoptotic effects on irradiated human UT-7/EPO cells through upregulation of Bcl-2 protein and the relative Bcl-2/Bax ratio, and by decreasing the activation of caspase 3. These findings may contribute to our understanding of the beneficial function of EPO in radiotherapy-induced anaemia.

The new targets of ouabain in retinal interneurons of Sprague-Dawley rats.

Ouabain is both a cardiac glycoside used in therapy of congestive heart failure and an endogenous steroid hormone. It specifically binds to Na(+), K(+)-ATPase (NKA) and blocks its activity. Overdose of ouabain induces retinal damage. In different species ouabain-induced retinal degeneration affects different cell types. In fish and rabbit ouabain induces retinal cell death preferentially in the ganglion cell layer and outer photoreceptor segments respectively. In rats, the pattern of NKA expression has been studied with most detail among retinal neurons. In addition, ouabain selectively destroyed some types of neurons in rodents. However, ouabain-sensitive retinal neurons remain unclear in rats. We show here that injection of ouabain into the rat vitreous body induced dramatic cell death in the inner nuclear layer (INL). The cell death was time- and dose-dependent. Ouabain-induced dying cells in the INL were TUNEL-positive. Immunohistochemistry analysis revealed that there was a significant decrease in the number of calbindin D-28K- and syntaxin-1-positive horizontal and amacrine cells in the INL of ouabain-treated rat retinas. Thus our results revealed that the horizontal and amacrine cells are the most sensitive cell types to ouabain in the retina of Sprague-Dawley rat.

Comparison of blood-nerve barrier disruption and matrix metalloprotease-9 expression in injured central and peripheral nerves in mice.

To investigate the involvement of blood-born factors and extracellular proteases in axonal degeneration and regeneration in both PNS and CNS, we directly compared the differences of blood-nerve barrier (BNB) disruption and matrix metalloprotease-9 (MMP-9) induction between the sciatic nerve and optic nerve after crush injury in the same animal. In sciatic nerve, BNB disruption, fibrin(ogen) deposition and MMP-9 expression were observed only in the first week following injury. Neurofilament (NF) immunoreactivity dramatically decreased in the first 2 days, gradually recovered to the normal levels by day 28. In contrast, the immunoglobulin G deposits spanned from 4 h to 28 days in crushed optic nerves. Fibrin(ogen) deposition was only observed in the first 2 days, while MMP-9 induction did not occur until a week after injury but lasted for 3 weeks in the crushed optic nerves. The NF immunoreactivity did not change much until day 7 and almost completely disappeared on day 28. The decrease of NF immunoreactivity coincided with the induction of MMP-9 after optic nerve crush. These results show that BNB disruption and MMP-9 induction are differentially regulated in the PNS and CNS after injuries, and they may contribute to the different regeneration capacities of the two systems.

Sonic hedgehog promotes stem-cell potential of Müller glia in the mammalian retina.

Müller glia have been demonstrated to display stem-cell properties after retinal damage. Here, we report this potential can be regulated by Sonic hedgehog (Shh) signaling. Shh can stimulate proliferation of Müller glia through its receptor and target gene expressed on them, furthermore, Shh-treated Müller glia are induced to dedifferentiate by expressing progenitor-specific markers, and then adopt cell fate of rod photoreceptor. Inhibition of signaling by cyclopamine inhibits proliferation and dedifferentiation. Intraocular injection of Shh promotes Müller glia activation in the photoreceptor-damaged retina, Shh also enhances neurogenic potential by producing more rhodopsin-positive photoreceptors from Müller glia-derived cells. Together, these results provide evidences that Müller glia act as potential stem cells in mammalian retina, Shh may have therapeutic effects on these cells for promoting the regeneration of retinal neurons.

Brain delivery property and accelerated blood clearance of cationic albumin conjugated pegylated nanoparticle.

Cationic bovine serum albumin (CBSA) conjugated poly(ethyleneglycol)-poly(lactide) (PEG-PLA) nanoparticle (CBSA-NP), was designed as a novel drug carrier for brain delivery. In this paper, three formulations of CBSA-NP with different surface CBSA density as well as native bovine serum albumin conjugated nanoparticle (BSA-NP) and CBSA unconjugated pegylated nanoparticle (NP), were formulated. Their brain transcytosis across the blood-brain barrier (BBB) coculture and brain delivery in mice were investigated using 6-coumarin as fluorescent probe. By using free CBSA as specific inhibitor, it was evidenced that CBSA-NP crossed the brain capillary endothelium through absorptive mediated transcytosis. The result of transcytosis across the BBB coculture and brain delivery in mice proved that the increase of surface CBSA density of the nanoparticle enhanced the BBB permeability-surface area but decreased blood AUC. The optimized CBSA number conjugated per averaged nanoparticle was 110, with the maleimide-PEG-PLA/methoxy-PEG-PLA weight ratio 1:10, which can acquire the greatest percentage of injected dose per gram brain (%ID/g brain) by 2.3-fold compared with NP. Besides, "accelerated blood clearance phenomenon" was found through evaluating blood clearance profile of CBSA-NP post-injection of single dose or over a period of successive high doses of CBSA-NP. Understanding these issues is important for the future development of CBSA-NP as a brain delivery carrier and for the attenuation of toxicity or immunological responses to the nanodevice following a consequence of nanomedication.

Aclarubicin-loaded cationic albumin-conjugated pegylated nanoparticle for glioma chemotherapy in rats.

Traditional glioma chemotherapy with those second-line drugs such as anthracyclines usually failed because they are inaccessible to blood-brain barrier (BBB) in tumor. In our study, we incorporated aclarubicin (ACL) into cationic albumin-conjugated pegylated nanoparticle (CBSA-NP-ACL) to determine its therapeutic potential of rats with intracranially implanted C6 glioma cells. When labeled with fluorescent probe, 6-coumarin, CBSA-NP was shown to accumulate much more in tumor mass than nanoparticle without conjugated CBSA (NP) 1 hr post intravenous injection, as well as better retention after 24 hr. Tumor drug concentration of CBSA-NP-ACL displayed 2.6- and 3.3-fold higher than that of NP-ACL and ACL solution 1 hr post injection, while 2.7 and 6.6-fold higher after 24 hr, respectively. Moreover, using tumor microdialysis sampling, AUC(0-24 hr) of free drug amount in tumor interstitium delivered by CBSA-NP-ACL was about 2.0- and 2.7-fold higher than that of NP-ACL and ACL solutions, respectively. When the tumor rat model was subjected to 4 cycles of 2 mg/kg of ACL in different formulations, a significant increase of median survival time was found in the group of CBSA-NP-ACL compared with that of saline control animals, animals treated with NP-ACL and ACL solution. By terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling, CBSA-NP-ACL can extensively make the tumor cell apoptosis. Histochemical evaluation by periodic acid Shiff staining and biochemical analysis depicted that the incorporation of ACL into CBSA-NP reduced its toxicity to liver, kidney and heart. Besides, CBSA-NP-ACL was not shown to open tight junction evaluated by BBB coculture. It was concluded that CBSA-NP-ACL could have a therapeutic potential for treatment of glioma.

Cationic albumin-conjugated pegylated nanoparticles allow gene delivery into brain tumors via intravenous administration.

Patients with malignant gliomas have a poor prognosis because these tumors do not respond well to conventional treatments. Studies of glioma xenografts suggest that they may be amenable to gene therapy with cytotoxic genes, such as the proapoptotic Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL). Gene therapy of gliomas ideally employs i.v. given vectors, thus excluding viral vectors as they cannot cross the brain microvascular endothelium or blood-brain barrier. Recently, we reported the synthesis of cationic albumin-conjugated pegylated nanoparticles (CBSA-NP) and showed their accumulation in mouse brain cells upon i.v. administration. In this study, plasmid pORF-hTRAIL (pDNA) was incorporated into CBSA-NP, and the resulting CBSA-NP-hTRAIL was evaluated as a nonviral vector for gene therapy of gliomas. Thirty minutes after transfection of C6 glioma cells, CBSA-NP-hTRAIL was internalized and mostly located in the cytoplasm, whereas NP-hTRAIL was entrapped in the endolysosomal compartment. At 6 and 48 hours after transfection, respectively, released pDNA was present in the nuclei and induced apoptosis. At 30 minutes after i.v. administration of CBSA-NP-hTRAIL to BALB/c mice bearing i.c. C6 gliomas, CBSA-NP-hTRAIL colocalized with glycoproteins in brain and tumor microvasculature and, via absorptive-mediated transcytosis, accumulated in tumor cells. At 24 and 48 hours after i.v. administration of CBSA-NP-hTRAIL, respectively, hTRAIL mRNA and protein were detected in normal brain and tumors. Furthermore, repeated i.v. injections of CBSA-NP-hTRAIL induced apoptosis in vivo and significantly delayed tumor growth. In summary, this study indicates that CBSA-NP-hTRAIL is a promising candidate for noninvasive gene therapy of malignant glioma.

Acute photoreceptor degeneration down-regulates melanopsin expression in adult rat retina.

Melanopsin in retinal ganglion cells plays an important role in mammalian circadian systems. Previous studies indicate melanopsin is responsible for circadian photoentrainment independent of classical rods and cones. However, expression of melanopsin in ganglion cells may be regulated by photoreceptors. In this study, we investigated the effects of N-methyl-N-nitrosourea (MNU)-induced acute photoreceptor degeneration on melanopsin mRNA expression and protein distribution in adult rats. Expression of melanopsin was analyzed 0.5, 1, 5, 7, 13 and 28 days after MNU administration by real-time RT-PCR and immunohistochemistry. MNU-induced gradual degeneration of photoreceptors, and by day 7 most of the photoreceptors were lost. The number of ganglion cells did not change significantly at all time points after MNU injection. In contrast, melanopsin mRNA decreased gradually with the loss of photoreceptors, at the same time pituitary adenylate cyclase-activating polypeptide (PACAP) mRNA levels, which co-express with melanopsin in ganglion cells, were not affected by MNU treatment, indicating decrease of melanopsin mRNA levels is not due to ganglion cell damage. Distribution of melanopsin protein in the dendrites of ganglion cells dramatically decreased with the degeneration of photoreceptors, but its expression in the soma persisted for a long time. Our results suggest that intact photoreceptors maintain the expression of melanopsin and its distribution in ganglion cell dendrites.