HBV X protein-based therapeutic vaccine accelerates viral antigen clearance by mobilizing monocyte infiltration into the liver in HBV carrier mice.

BACKGROUND: Hepatitis B virus (HBV) persistently infected about 250 million people worldwide, and a curative treatment remains an unmet medical need. Among many approaches to treat chronic hepatitis B (CHB), therapeutic vaccines have been developed for two decades, but none have yielded promising results in clinical trials. Therefore, dissection of HBV clearance mechanisms during therapeutic vaccination in appropriate models, which could give rise to new curative therapies, is urgently needed. Growing evidence indicates that prolonged and intensive exposure of antigen-specific T cells to viral antigens is a major cause of T cell exhaustion, and decreases anti-HBV immunity efficacy of therapeutic vaccination. HBV X protein (HBx) is expressed at low levels, and the understanding of its immunogenicity and potential in therapeutic CHB vaccines is limited. METHODS: HBV genome sequences from CHB patients were cloned into a pAAV plasmid backbone and transfected into immunocompetent mouse hepatocytes through hydrodynamic injection. Mice carrying > 500 IU/mL serum HBV surface antigen (HBs) for more than 4 weeks were considered HBV carriers mimicking human CHB and received 3 doses of weekly HBx vaccine by subcutaneous immunization. Serum HBV clearance was evaluated by monitoring serum HBs and HBV-DNA titers. Residual HBV in the liver was evaluated by western blotting for HBV core antigen. The splenic antigen-specific T cell response was quantified by a 15-mer overlapping peptide-stimulated interferon-γ enzyme-linked immunospot assay. Blood and hepatic immune cells were quantified by flow cytometric analysis. RESULTS: Our HBx-based vaccine induced systemic HBx-specific CD4+ and CD8+ T cell responses in HBV carrier mice and demonstrated significant HBs and HBV-DNA elimination. The protective effect persisted for at least 30 days without additional booster immunization. Different infiltrating myeloid cell subsets, each with distinctive roles during immune-mediated HBV clearance, were found in the liver of vaccinated mice. During vaccine therapy, inflammatory monocyte depletion resulted in sustained HBV clearance inhibition, whereas phagocytic monocyte-derived macrophage and Kupffer cell elimination resulted in only transient inhibition of vaccine-induced HBV clearance. CONCLUSIONS: We report the potential role of HBx as a major immunogen in an HBV therapeutic vaccine and the significance of a liver-infiltrating monocyte subset during hepatic viral clearance.

Electrospray Synthesis of Poly(lactide-co-glycolide) Nanoparticles Encapsulating Peptides to Enhance Proliferation of Antigen-Specific CD8+ T Cells.

Polymer nanoparticles (NP) are of escalating interest for their application as immune stimulatory pharmaceutics. The production of nanosized carrier systems is currently being widely investigated, but commonly used techniques, such as the double emulsion technique, are limited by shortcomings of low encapsulation efficiency and poor control over size distribution. In this study, the electrospray technique was successfully implemented and optimized to produce monodisperse 200-nm poly(lactide-co-glycolide) (PLGA) NP. For cytomegalovirus (CMV) pp65 and IE-1 peptides, a consistent encapsulation efficiency of approximately 85% was achieved. In vitro stimulation of peripheral blood mononuclear cells (PBMCs) from CMV+ donors using electrosprayed pp65489-503 peptide-loaded NP revealed a significantly increased proliferation rate and frequency of antigen-specific CD8+ T cells as compared to the soluble peptide. The results of this study demonstrate the suitability of the electrospray technique for production of monodisperse PLGA NP with high drug encapsulation efficiency as promising peptide-based vaccine carriers.

[Preliminary study on transdermal characteristics and sunface anesthetic effects of lidocaine hydrochloride loaded trans-activator of transcription peptide conjugated nano-niosome in animals].

OBJECTIVE: To prepare a new dental topical anesthetics, lidocaine hydrochloride loaded trans-activator of transcription peptide conjugated nano-niosome (LID-TAT-N), and to evaluate its transdermal properties and topical anesthesia effects. METHODS: LID-TAT-N was prepared using reverse-phase evaporation method, and lidocaine loaded conventional liposome (LID-CL) was prepared in the same manner as positive control. The diameter, ζ potential and encapsulation efficiency of LID-TAT-N and LID-CL were measured. The skin permeation of LID-TAT-N was examined, and compared with LID-CL and lidocaine injection (LID-IJ, as negative control), using a Franz diffusion cell mounted with depilated mouse skin in vitro for 12 hours. Each experiment was repeated six times. The anesthetic effect of the new topical anesthetic was investigated on the cornea of rabbits. RESULTS: The mean diameter of LID-TAT-N was smaller than that of LID-CL [(152.7 ± 10.6) nm vs. (259.5 ± 15.5) nm, P < 0.01]. The 12 h cumulative permeation amount was significantly higher in LID-TAT-N group [(1 340.0 ± 97.5) µg · cm(-2)] than those of LID-CL and LID-IJ groups [(1 060.6 ± 80.2), (282.6 ± 65.1) µg · cm(-2), respectively, P < 0.05]. Rabbit corneal reflex results showed that LID-TAT-N had anesthetic effect and the duration of analgesia [(24.8 ± 2.8) min] was also longer than that of LID-IJ [(14.5 ± 2.3) min, P < 0.05]. CONCLUSIONS: LID-TAT-N had good transdermal ability, and the advanced skin penetration feature can improve its tropical anesthetic effect.

Oral administration of PDX1 confers protection against insulitis in the non-obese diabetic (NOD) mice.

Type 1 diabetes is a T cell-mediated organ-specific autoimmune disease. Antigen-specific immune intervention allows the selective targeting of autoreactive T cell, while leaving the remainder of the immune system intact. However, immune intervention for type 1 diabetes has not yielded perfect results clinically. In our paper published previously, we asked whether pancreatic duodenal home box 1 (PDX1) is a target of anti-islet autoimmunity in type 1 diabetes. In this experiment, we assessed the therapeutic effect of oral administration of PDX1 on diabetes development of 4-week-old non-obese diabetic (NOD) mice. The results indicate that PDX1 immunization is an effective intervention strategy for delaying the onset of diabetes in NOD mice in association with: 1) reduced insulitis; 2) suppression of destructive autoreactive T cells; 3) augmentation of regulatory T cells; 4) a shift in cytokine production. The present observations suggest that immunization with PDX1 modulates immune cell responses in NOD mice, raising the possibility that it is beneficial in ameliorating autoimmune destruction of beta-cells and delaying type 1 diabetes development clinically.

Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo.

Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells.

Cooperation of myocardin and Smad2 in inducing differentiation of mesenchymal stem cells into smooth muscle cells.

Several reports demonstrated that mesenchymal stem cells (MSCs) might differentiate into smooth muscle cells (SMCs) in vitro and in vivo. It has been shown that myocardin protein is a strong inducer of smooth muscle genes and MSCs can differentiate into SMCs in response to transforming growth factor-ß (TGF-ß). However, the relationship or link between myocardin and TGF-ß3-induced MSC differentiation has not been fully elucidated. Here, we demonstrated that both myocardin and TGF-ß3 were able to induce differentiation of rat bone marrow-derived MSCs toward smooth-muscle-like cell types, as evidenced by increasing expression of SMC-specific genes. Of note, myocardin cooperated with Smad2 to synergistically activate SM22α promoter and significantly enhance the expression of SM22α. Report assays with site-direct mutation analysis of SM22α promoter demonstrated that myocardin and Smad2 coactivated SM22α promoter mainly depending on CArG box and less on smad binding elements (SBE) sites as well. These findings reveal the cooperation of myocardin and Smad2 in process of MSC differentiation into SMCs.

Reversal of streptozotocin-induced diabetes in mice by cellular transduction with recombinant pancreatic transcription factor pancreatic duodenal homeobox-1: a novel protein transduction domain-based therapy.

OBJECTIVE: The key pancreatic transcription factor pancreatic duodenal homeobox-1 (Pdx1), known to control development and maintenance of pancreatic beta-cells, possesses a protein transduction domain (PTD) that facilitates its entry into cells. We therefore sought to evaluate the capacity of in vivo-administered recombinant Pdx1 (rPdx1) to ameliorate hyperglycemia in mice with streptozotocin-induced diabetes. RESEARCH DESIGN AND METHODS: Cell entry and transcriptional regulatory properties of rPdx1 protein and its PTD-deletion mutant rPdx1Delta protein, as well as a PTD-green fluorescent protein, were evaluated in vitro. After intraperitoneal rPdx1 injection into mice with streptozotocin-induced diabetes, we assessed its action on blood glucose levels, insulin content, intraperitoneal glucose tolerance test (IPGTT), Pdx1 distribution, pancreatic gene expression, islet cell proliferation, and organ histology. RESULTS: Restoration of euglycemia in Pdx1-treated diabetic mice was evident by improved IPGTT and glucose-stimulated insulin release. Insulin, glucagon, and Ki67 immunostaining revealed increased islet cell number and proliferation in pancreata of rPdx1-treated mice. Real-time PCR of pancreas and liver demonstrated upregulation of INS and PDX1 genes and other genes relevant to pancreas regeneration. While the time course of beta-cell gene expression and serum/tissue insulin levels indicated that both liver- and pancreas-derived insulin contributed to restoration of normoglycemia, near-total pancreatectomy resulted in hyperglycemia, suggesting that beta-cell regeneration played the primary role in rPdx1-induced glucose homeostasis. CONCLUSIONS: rPdx1 treatment of mice with streptozotocin-induced diabetes promotes beta-cell regeneration and liver cell reprogramming, leading to restoration of normoglycemia. This novel PTD-based protein therapy offers a promising way to treat patients with diabetes while avoiding potential side effects associated with the use of viral vectors.

Increase of proliferating oligodendroglial progenitors in the adult mouse brain upon Sonic hedgehog delivery in the lateral ventricle.

Sonic hedgehog signaling is required for the maintenance of stem cell niches in the postnatal subventricular zone and the proliferation of neural progenitors in the mature hippocampus. We show here that delivery of Sonic hedgehog protein into the lateral ventricle of adult mice increases cell proliferation in the corpus callosum and cerebral cortex. In this latter area, the number of neural progenitors expressing the proteoglycan NG2 is enhanced 2 days after the injection. In both areas, mRNA up-regulation of the transcriptional target gene Patched was observed in cells expressing the oligodendroglial transcription factor Olig1. Twenty-six days following the adenovirus-mediated delivery of Sonic hedgehog into the lateral ventricle, newly generated cells in the cerebral cortex and in the corpus callosum are influenced towards the initial steps of oligodendrogenesis, as indicated by a 50% increase in the number of cells expressing the oligodendroglial marker DM20. Our experiments demonstrate that the number of oligodendrocyte precursor cells in the cerebral cortex and corpus callosum can be increased upon delivery of Sonic hedgehog proteins and highlight the potential capacity of the adult brain to mobilize a pool of premyelinating cells.

Sonic hedgehog stimulates the proliferation of rat gastric mucosal cells through ERK activation by elevating intracellular calcium concentration.

Sonic Hedgehog (Shh), a member of hedgehog peptides family, is expressed in gastric gland epithelium. To elucidate Shh function to gastric mucosal cells, we examined the effect of Shh on the proliferation of a rat normal gastric mucosal cell line, RGM-1. RGM-1 cells express essential components of Shh receptor system, patched-1, and smoothened. Shh enhanced DNA synthesis in RGM-1 cells and elevated intracellular calcium concentration ([Ca2+]i). In addition, Shh as well as calcium ionophore A32187 rapidly activated ERK. However, Shh failed to activate ERK under calcium-free culture condition. Pretreatment of cells with PD98059 attenuated the DNA synthesis promoted by Shh. Moreover, when cells were pretreated with cyclopamine, Shh could not elevate [Ca2+]i, activate ERK or promote DNA synthesis. On the other hand, although Shh induced Gli-1 nuclear accumulation in RGM-1 cells, Shh activated ERK even in cells pretreated with actinomycin D. These results indicate that Shh promotes the proliferation of RGM-1 cells through an intracellular calcium- and ERK-dependent but transcription-independent pathway via Patched/Smoothened receptor system.

A novel approach to thymic rejuvenation in the aged.

With advancing age, the mammalian thymus undergoes involution, a progressive loss of architectural integrity and lymphoid cellularity that results in reduced T lymphopoiesis. Thymic involution also is associated with extreme malnutrition and states of immune deficiency, such as active HIV infection, after chemotherapy, or during pregnancy. Immune recovery appears to require restoration of normal thymopoiesis. Although several means are known to increase thymic cellularity in the aged, including systemic administration of hormones, androgen ablation, and thymic tissue transplantation, each suffers from specific limitations that prevent widespread application. This paper presents a novel approach to rejuvenate T cell differentiation in the aged that employs intrathymic implantation of engineered stromal cells. Two different proteins have been examined for their impact on thymopoiesis after delivery by somatic cell implantation. Intrathymic injection of IL-7-producing stromal cells enhances the earliest specification steps of T cell development, resulting in the increased representation of pro-T cells in the aged thymus. In contrast, increasing the intrathymic levels of sonic hedgehog diminishes this aspect of T cell poiesis.

Immunization with antigen-pulsed dendritic cells significantly improves the immune response to weak self-antigens.

Dendritic cells (DCs) are the only professional antigen-presenting cells endowed with the ability to stimulate naïve T cells and initiate a primary immune response. For this reason, DC-based immunization has been shown to be highly effective in eliciting CTL responses to viruses and tumor-associated antigens. Here we report on the use of DC immunization to enhance the B cell-mediated humoral immune response to highly conserved proteins and the application of this approach to the generation of monoclonal antibodies (mAbs) against these proteins. To illustrate the technique we describe the production of mAbs to class II transactivator (CIITA), the major histocompatibility complex (MHC) CIITA, a difficult immunogen owing to its high degree of identity among species. We show that mice immunized with a combination of an intravenous injection of DCs pulsed with recombinant fragments of CIITA followed by intraperitoneal injection of the antigen in incomplete Freund's adjuvant induced a detectable antibody response against CIITA, while sera from mice immunized using the traditional method (i.e. intraperitoneal immunization with 50mug of protein in complete Freund's adjuvant) gave an almost undetectable response. Furthermore, a total of four fusion experiments demonstrate that immunization with Ag-pulsed DCs is necessary for the efficient generation of hybridomas and a good yield of mAbs specific for the recombinant and the native endogenous CIITA protein. Conversely, four independent fusions carried out with splenocytes from mice immunized using the traditional method failed to produce anti-CIITA hybridomas. We propose that immunization with antigen-loaded DCs should be the method of preference when attempting to raise mAbs against weak self-immunogens.

Cytological aspects of rat mammary cancer therapy with soluble tumor-associated antigens and anticancer drugs.

Some cytological aspects of rat mammary cancer therapy with soluble tumor-associated antigens (sTAA- p66 and p51) and the anticancer drug cyclophosphamide (CPA) are analyzed. Vaccination with sTAA results in a significant increase in the areas related to the production of T and B cells in the white pulp (germinal center and PALS) and in the marginal zone of the spleen. sTAA stimulate the production of CD8+ lymphocytes inside the tumors and in bone marrow, and of CD8+ thymocytes in the medulla of the thymus. Treatment of rats with CPA decreases the activity of lymph cells in tumors, especially of CD4+ lymphocytes. In the spleen, CPA decreases the size of areas related to the production of B and T cells. In the bone marrow, CPA affects the process of myelogenesis and causes significant substitution of cellular components with fatty tissue. The combined treatment with CPA and sTAA increases the number of lymph cells and the apoptotic index in tumors, and restored the rate of B cells producing in the spleen. Similar effect was observed in lymph nodes with accumulation of B lymphocytes in the primary and secondary follicles, and of T lymphocytes in the paracortical zone. In the thymus, CPA alone or in combination with sTAA repairs the inhibitor effect of a carcinogen on synthesis of CD4+ and CD8+ thymocytes. In combined using with CPA, sTAA activate the B- and T-lymphocyte production in the host's immune system and decrease the toxic side-effects of a drug.

Intravenous delivery of liposome-mediated nonviral DNA is less toxic than intraperitoneal delivery in mice.

Suicide gene therapy has been shown to be an effective means of destroying pancreatic cancer cells. Liposomes have been described as having better efficacy in gene delivery, and an advantage of using liposomes as gene carriers is that they can be used repeatedly in vivo. The objective of this study is to compare the effect of gene delivery routes and to determine whether systemic delivery of the rat insulin promoter (RIP)-directed suicide gene construct would permit cell-specific gene delivery in vivo. Severe combined immunodeficient (SCID) mice were injected with liposome-RIP-TK (thymidine kinase) complex by either the intraperitoneal or the intravenous route. Twenty-four hours post gene delivery, mice received ganciclovir (GCV) treatment twice daily for 14 days. Mice were sacrificed at various time points. Complete necropsy and serum chemistry analysis were performed. Islet morphology was determined using hematoxylin and eosin (H&E) staining. Serum glucose and insulin levels were also determined. To determine the toxic effect on pancreatic islet cells, immunostaining of insulin-producing and glucagon-producing cells was carried out at each time point. H&E staining indicated that both intravenous and intraperitoneal liposome-RIP-TK gene expression had no effect in normal endocrine islet cells. Both gene-delivery routes in mice resulted in normal glycemia and serum insulin levels. The endocrine islets were intact, with a normal distribution pattern of insulin-producing beta cells and glucagon-secreting alpha cells. However, serum chemistry analysis revealed significantly elevated levels of liver enzymes; suggesting that possible liver damage had occurred with the intraperitoneal gene delivery of liposome-pRIP-TK. Intravenous liposome-mediated gene delivery had no effect on liver enzyme levels. Liposome-mediated gene delivery via intravenous injection was less toxic than intraperitoneal delivery. This gene-delivery route requires fewer liposome-DNA complexes and maintains normal liver function. Thus, intravenous delivery of gene therapy would be superior to intraperitoneal administration of gene therapy in mice.

Sonic hedgehog delivered by an adeno-associated virus protects dopaminergic neurones against 6-OHDA toxicity in the rat.

Direct intracerebral administration of sonic hedgehog (SHH) reduces 6-OHDA and MPTP toxicity to nigral dopaminergic cells in rats and primates. To determine whether transfection of the DNA sequence for SHH using viral vectors also protects against 6-OHDA toxicity, a type 2 adeno- associated virus (AAV) incorporating 600 base pairs of N-terminal SHH DNA was generated to induce SHH expression in rat striatum.AAV-SHH was injected into the striatum, 3 weeks prior to the initiation of an unilateral partial 6-OHDA nigro-striatal lesion. Animals receiving 4x10(7) viral particles of AAV-SHH showed a reduction in (+)-amphetamine induced ipsilateral turning over 4 weeks, when compared to animals receiving vehicle or a LacZ encoding vector. Following vehicle or AAV-LacZ administration, 6-OHDA caused a marked loss of striatal dopamine content and nigral tyrosine hydroxylase (TH) immunopositive cells. Following treatment with 4x10(7) viral particles of AAV-SHH the loss of striatal dopamine content was reduced and there was marked preservation of nigral dopaminergic cells. However, administration of 4x10(8) particles of AAV-SHH did not cause a significant change in (+)-amphetamine-induced rotation, striatal dopamine levels or the number of nigral TH immunoreactive cells following 6-OHDA lesioning compared to vehicle or AAV-LacZ treated animals. The results show that SHH delivered via a viral vector can protect dopaminergic neurons against 6-OHDA toxicity and suggest that this could be developed into a novel treatment for PD. However, the effects maybe dose limited due to uncoupling of hedgehog receptor signalling at higher levels of SHH expression.

Oral immunization with an rfaH mutant elicits protection against salmonellosis in mice.

Loss of the transcriptional antiterminator RfaH results in virulence attenuation (>10(4)-fold increase in 50% lethal dose) of the archetypal Salmonella enterica serovar Typhimurium strain SL1344 by both orogastric and intraperitoneal routes of infection in BALB/c mice. Oral immunization with the mutant efficiently protects mice against a subsequent oral infection with the wild-type strain. Interestingly, in vitro immunoreactivity is not confined to strain SL1344; rather, it is directed also towards other serovars of S. enterica and even Salmonella bongori strains.

Relationship between NMDA receptor expression and MPP+ toxicity in cultured dopaminergic cells.

It has been suggested that excitotoxicity could be contributing to dopamine cell loss after methylphenylpyridinium ion (MPP+) exposure, although the literature regarding this is contradictory. Given that in cell culture excitotoxicity has been reported to be dependent on culture age, we postulated that these discrepant results might be explained by a difference in developmental expression of N-methyl-D-aspartate (NMDA) receptors. To test this, mesencephalic cells were cultured and the number of dopaminergic neurons (tyrosine hydroxylase-immunoreactive cells [TH-IR] cells) expressing the NMDA R1 subunit (NR1) was determined using double-label immunofluorescence microscopy. An increase in the percentage of TH-IR cells expressing NR1 occurred over time in culture and this correlated with the toxicity of NMDA. At 7 days in vitro (DIV 7), only 17% (n=167 cells/4 experiments) of TH-IR cells expressed NR1 and these cells were insensitive to NMDA toxicity. This increased to 80% (n=254 cells/6 experiments) by DIV 11 and cultures were now susceptible to NMDA-induced injury. Cultures grown for either 7 or 11 days were treated for 48 hr with increasing concentrations of MPP= (0.5-20 microM) and the loss of dopaminergic neurons was determined by cell counting. Cultures at DIV 7 were more sensitive to MPP= than 11-day-old cultures (LD50= approximately 0.75 microM vs. 15 microM, respectively). Co-exposure to MK-801 (5 microM) did not protect against MPP+ toxicity in young cultures, but attenuated MPP+ toxicity in the older cultures, becoming statistically significant at 20 microM MPP+. These data indicate that the activation of NMDA receptors is not required for, but can contribute to, MPP(+)-induced neurodegeneration of dopaminergic cells in culture.

Sonic hedgehog-induced neural precursor proliferation after adult rodent spinal cord injury.

OBJECT: The glycoprotein molecule sonic hedgehog (Shh) has been shown to play a critical role in neuraxial development. To assess its role in the repair of demyelination following spinal cord injury (SCI), escalating doses of Shh were injected into demyelinated lesions in adult rat spinal cords. METHODS: Twenty-seven adult rats underwent thoracic laminectomy and chemical demyelination of the spinal cord dorsal columns without neurological deficit A subset of 20 rats was treated after 3 days by direct injection of Shh at two different doses. Rats were killed at 7 or 21 days after SCI, and tissue samples underwent immediate fixation or were placed into cell culture. Diffuse cellular proliferative responses throughout the gray and white matter were observed in up to 70% of Shh-treated rats. Proliferation around the central canal, believed to be derived from the ventricular ependyma consistent with neuronal stem cell induction, was demonstrated in up to 60% of the treated rats. No significant proliferation in these areas was detected in control rats. Dorsal areas of nestin-positive cells were also observed in 70% of rats treated with high doses of Shh, and these observations were reproduced in cell culture as well as in cultures of dorsal spinal cord explants. Cell counts revealed significant increases in the percentage of oligodendrocyte precursors and neurons in treated compared with control rats. CONCLUSIONS: Exogenous Shh administration promotes nestin-positive cell proliferation after SCI in adult rodents. These cells are believed to be derived from neural precursor cells. The populations of oligodendrocyte precursors and neurons were likewise increased in Shh-treated rats, suggesting that these cells may be derived from neural stem cells.

Regression of a mammary adenocarcinoma in STAT6-/- mice is dependent on the presence of STAT6-reactive T cells.

Polarization of the immune response toward a type 1 cytokine profile has been posited to be associated with a therapeutic antitumor immune response. STAT6-/- mice are unable to generate a type 2 immune response, and instead mount an enhanced type 1 response. STAT6-/- mice are significantly more resistant to 4T1, a mammary adenocarcinoma cell line, resisting a 10-fold higher tumor dose compared with wild-type (wt) BALB/c mice. An analysis of the T cells from tumor-bearing STAT6-/- mice revealed that they contained a population primed by a peptide (STAT6(531-539)) of the STAT6 protein expressed in 4T1. The adoptive transfer of T cells from STAT6(531-539)-vaccinated STAT6-/- mice significantly reduced the number of 4T1 pulmonary metastases in recipient mice. Additionally, the role of these STAT6(531-539)-reactive T cells against s.c. 4T1 tumor challenge was determined by tumor-challenging wt BALB/c mice reconstituted with STAT6-/- bone marrow, thereby assessing whether a polarized type 1 immune response in the absence of STAT6-reactive T cells was sufficient to reject a 4T1 tumor challenge. T cells from the STAT6-/- bone marrow chimeras failed to recognize the STAT6(531-539), and these mice proved to be as susceptible as wt BALB/c mice to 4T1 challenge. This demonstrated that the absence of STAT6(531-539)-reactive T cells correlated with the inability to reject 4T1 challenge. Additionally, these data emphasize that the enhanced ability to mount a type 1-polarized immune response is inconsequential if a sufficient antitumor immune response is not primed by the tumor.

Tumor eradication by hepatitis B virus X antigen-specific CD8+ T cells in xenografted nude mice.

We have previously reported several CTL epitopes derived from the hepatitis B viral X Ag (HBx). In this study, we evaluated whether HBx-specific CTLs can be effectively used in adoptive cancer immunotherapy. To validate the possibility, four peptides containing a HLA-A2.1-restricted binding consensus motif were identified from the HBx protein and tested for their ability to activate CTL from PBMCs isolated from chronic carriers of HBV (n = 12). We selected two highly potent epitopes, HBx 52-60 (HLSLRGLFV) and HBx 115-123 (CLFKDWEEL), that are capable of inducing Ag-specific cytotoxic T cells in patient PBMCs. For adoptive immunotherapy using HBx-specific CTLs, we generated CTL clones restricted to the HBx 52-60 or HBx 115-123 peptide using a limiting dilution technique. LC-46, an HBx 52-60-specific clone, is CD62L(-)CD69(+)CD45RO(+)CD45RA(-)CD25(dim) and is stained by IFN-gamma (approximately 92%), IL-2 (30%), and TNF-alpha (56%), but not by IL-5, IL-10, IL-12, or TNF-beta, indicating that the cells are fully activated T cytotoxic 1-type cells. When LC-46 cells were adoptively transferred into xenografted nude mice bearing human hepatomas expressing HLA-A2.1 molecules and intracellular HBx proteins, the tumors were eradicated. Taken together, our data provide solid evidence for the feasibility of adoptive immunotherapy with HBx-sensitized CTLs in hepatitis disease, including hepatocellular carcinoma (HCC).

Intrastriatal injection of sonic hedgehog reduces behavioral impairment in a rat model of Parkinson's disease.

Sonic hedgehog (Shh), a member of hedgehog (hh) family of signaling molecules, is necessary for normal axial patterning and cellular differentiation in the developing central nervous system. Shh also promotes the survival of fetal dopaminergic neurons and protects cultures of fetal midbrain dopaminergic neurons from the toxic effects of N-methyl-4-phenylpyridinium (MPP(+)), a neurotoxin that selectively injures nigral dopaminergic neurons. The mRNA expression of Shh and its putative receptor in the adult brain indicates an important role of Shh in the mature nervous system in addition to its roles during embryogenesis. In this study we examined the behavioral and anatomical effects of intrastriatal injection of singly myristoylated wild-type human Sonic hedgehog N-terminal fragment (Shh-M) in a rat model of Parkinson's disease (PD). Five groups of rats received a series of four intrastriatal injections of Shh-M (180 ng, 540 ng, or 4.275 microg per injection), glial cell line-derived neurotrophic factor (GDNF) (1 microg/injection), or vehicle on days 1, 3, 5, and 8. On day 4, the animals received an intrastriatal injection of 15 microg 6-hydroxydopamine (6-OHDA) free base. Intrastriatal administration of Shh (180 ng/injection) twice before and after a single intrastriatal injection of 6-OHDA reduced apomorphine- and amphetamine-induced rotation and forelimb akinesia and partially preserved dopaminergic axons in the striatum. This is the first demonstration in vivo that Shh reduces behavioral deficits induced by intrastriatal 6-OHDA lesion and suggests that Shh may be useful in the treatment of disorders that affect the nigrostriatal system, such as PD.