CD8(+) suppressor T cells (Ts) have been demonstrated to provide protection of allograft from rejection. We previously reported that soluble peptide/HLA-A2 dimer shows peptide-specific inhibitory effects on alloresponse in a coculture of peptide-pulsed T2 cells with HLA-A2 negative lymphocytes in vitro. Here, we found a subset of CD8(low)CD28(-) T cells was induced in the dimer-treated coculture. Importantly, this population showed hyporesponsiveness to the alloantigen restimulation as well as alloantigen-specific suppression on alloreactive T cells in a cell-cell contact-dependent fashion. The suppressive mechanisms of CD8(low)CD28(-) T cells involved an elevated expression of membrane-bound TGF-?1, but not Foxp3, CTLA-4 or IL-10. Furthermore, an overrepresention of CD8(low)CD28(-) T cells was observed in the patients after allogeneic platelet transfusion, and positively correlated with the elevated concentrations of plasma HLA class I antigens. Our findings demonstrated that soluble HLA-A2 dimer could efficiently induce the tolerant CD8(low)CD28(-) T cells with alloantigen-specific suppression on alloreactive T cells. This study might provide a new strategy for preparation of donor-specific Ts and represent an attractive alternative for induction of allograft tolerance.
Accumulative evidence has demonstrated that inflammation has an important role in human papillomavirus (HPV) oncogenicity. However, the effects of high mobility group protein box 1 (HMGB1)-toll like receptor 4 (TLR4) signaling pathway associated inflammation on epidermal warts caused by HPV remain unclear. The present study investigated the HMGB1, TLR4 and nuclear factor-?B p65 expression in condyloma acuminatum (CA) and verruca vulgaris (VV). Immunohistochemistry and western blot analysis revealed that p65 expression in epithelial nuclei in VV and CA was significantly higher than in normal skin (NS) (P<0.01), and p65 in CA was higher than in VV but this difference was not significant. The level of extracellular HMGB1 increased significantly and progressively from NS to VV to CA (P<0.05). The level of TLR4 on the surface of epithelial membranes in the CA samples was significantly higher than in NS (P<0.01), and TLR4 in VV samples was significantly lower than in NS (P<0.01). There was a positive correlation between p65 expression in the epithelial nuclei and HMGB1 in the epithelial intercellular spaces (r=0.5199, P<0.01). These findings indicate that inflammation is intensified in warts caused by HPV. HMGB1-TLR4 pathway-associated inflammation may therefore have a pivotal role in CA. HMGB1, rather than TLR4, may be a vital mediator of inflammation in VV. Therapies targeting HMGB1 may be a potential strategy for the treatment of HPV-associated warts.
Neutrophils and monocytes/macrophages (MMs) play important roles in the development of cell-mediated delayed type hypersensitivity (DTH). However, the dynamics of neutrophils and MMs during the DTH reaction and how the immunosuppressant rapamycin modulates their behavior in vivo are rarely reported. Here, we take advantage of multi-scale optical imaging techniques and a footpad DTH reaction model to non-invasively investigate the dynamic behavior and properties of immune cells from the whole field of the footpad to the cellular level. During the classic elicitation phase of the DTH reaction, both neutrophils and MMs obviously accumulated at inflammatory foci at 24 h post-challenge. Rapamycin treatment resulted in advanced neutrophil recruitment and vascular hyperpermeability at an early stage (4 h), the reduced accumulation of neutrophils (> 50% inhibition ratio) at 48 h, and the delayed involvement of MMs in inflammatory foci. The motility parameters of immune cells in the rapamycin-treated reaction at 4 h post-challenge displayed similar mean velocities, arrest durations, mean displacements, and confinements as the classic DTH reaction at 24 h. These results indicate that rapamycin treatment shortened the initial preparation stage of the DTH reaction and attenuated its intensity, which may be due to the involvement of T helper type 2 cells or regulatory T cells.
Three heavy-chain and three kappa (?)-chain transcripts were amplified from hybridoma cells secreting a monoclonal antibody (mAb) against transferrin receptor. Sequence analysis via IMGT/V-QUEST yielded the functional/aberrant prediction. Two functional ?-chain transcripts, V?2 and V?3, and one functional VH1 were revealed. Comprehensive bioinformatics analyses including sequence alignment, phylogenetic tree, somatic hypermutation prediction, and three-dimensional-molecular structure modeling were used to predict the origin of the two ?-chain transcripts. The results of bioinformatics analysis suggest that V?3 is derived from the myeloma partner of the hybridoma; V?2 is derived from B-cell. Functional transcripts VH1 and V?2 and V?3 were then used to construct two chimeric antibodies chi-C2 (V?2-VH1) and chi-C3 (V?3-VH1), respectively. Antigen-binding experiments showed that only chi-C2 remained the same affinity as its parental mAb. Possible explanations for the coexistence of two functional ?-chain transcripts and the different affinity of the two chimeric antibodies are discussed.
Tumor cells commonly adapt survival strategies by downregulation or mutational inactivation of TGF-? receptors thereby reversing TGF-?1-mediated growth arrest. However, TGF-?1-triggered signaling also has a protumor effect through promotion of tumor cell migration. The mechanism(s) through which malignant cells reconcile this conflict by avoiding growth arrest, but strengthening migration remains largely unclear. TGF-?RII was overexpressed in the bladder cancer cell line T24, concomitant with point mutations, especially the Glu269 to Lys mutation (G ? A). Whilst leaving Smad2/3 binding unaffected, TGF-?RII mutations resulted in the unaffected tumor cell growth and also enhanced cell mobility by TGF-?1 engagement. Such phenomena are perhaps partially explained by the mutated TGF-?RII pathway deregulating the p15 and Cdc25A genes that are important to cell proliferation and CUTL1 gene relevant to motility. On the other hand, transfecting recombinant TGF-?RII-Fc vectors or smad2/3 siRNA blocked such abnormal gene expressions. Clinically, such G ? A mutations were also found in 18 patients (n=46) with bladder cancer. Comparing the clinical and pathologic characteristics, the pathologic T category (?2 trend = 7.404, P<0.01) and tumor grade (?2 trend = 9.127, P<0.01) tended to increase in the G ? A mutated group (TGF-?RII point-mutated group). These findings provide new insights into how TGF-?1 signaling is tailored during tumorigenesis and new information into the current TGF-?1-based therapeutic strategies, especially in bladder cancer patient treatment.
This study aims to review clinical features, treatments, and prognostic factors of thrombotic thrombocytopenic purpura (TTP) associated with systemic lupus erythematosus patients (sTTP). The case reports of sTTP published in world literature from 1999 to 2011 were collected, and 105 cases were divided into death group and survival group. The epidemiologic characteristics, clinical manifestations, laboratory examinations, treatments, and prognostic factors were analyzed. We found that coexistence of renal and neurological impairments were significantly frequent in the death group (100 %) than in the survival group (56.5 %) (P?=?0.002). Type IV was predominant in 57.7 % of renal pathological damage, followed by type V (11.5 %), type II (5.8 %), and thrombotic microangiopathy (TMA) (5.8 %). TMA appeared more frequently (50 %) in the death group than in the survival group (6.25 %) (P?=?0.042). End-stage renal disease occurred in nine cases with type IV in five (55.6 %), type TMA in one (11.1 %), and unspecified in three cases (33.3 %). Of 32 cases, 40.6 % showed severe ADAMTS13 deficiency and returned to normal or mildly deficient after remission. The total mortality rate of sTTP was 12.4 % and the mortality rate of patients with infection (27.3 %) was significantly higher than those without infection (8.4 %) (P?=?0.028). Plasma exchange and glucocorticoids were administrated in over 80 % of cases with 65.7 % remission rate, while additional cytotoxics or rituximab was mostly used in refractory sTTP and achieved over 90 % of remission rate. Above all, coexistence of renal and neurological impairments, infection, and renal damage with type IV or TMA might denote a poor prognosis of sTTP.
Recent studies suggest that CD8(+) T cells with down-regulated CD8 expression (CD3(+)CD8(low) T cells) represented as a distinct phenotype of CD8(+) T cells are increased and linked to disease severity in some chronically persistent infection, such as chronic HIV and parasite infection. However, the role of CD3(+)CD8(low) T cells in the context of chronic HBV infection is poorly understood. In this study, peripheral blood samples of 47 chronic hepatitis B patients and 19 healthy controls were collected and tested for the frequency and phenotype of CD8(low) T cells. The circulating CD8(low) T cells were significantly more frequent in the patients compared to those in healthy controls, and the CD8(low) T cells in the patients expressed less IFN-? and more mTGF-?1 than those in the controls, suggesting their type-2 polarized and suppressive properties. Meanwhile, the concentrations of plasma soluble HLA class I molecules were found elevated in the patients, and positively associated with the frequencies of CD8(low) T cells. Furthermore, the CD8(low) T-cell frequency in the HLA-A2-positive patients (n=21) was found negatively correlated with the T-cell responsiveness against the HBc????? peptide, the latter was impaired as revealed by IFN-? Elispot assay. Our findings suggested that a better understanding of the involvement of CD8(low) T cells in chronically persistent HBV infection would add to our knowledge of the impaired T-cell response in the patients.
The hypoxia-inducible factor-1 (HIF-1?) pathway is associated with tumor growth, angiogenesis and metastasis in various carcinomas. Little is known regarding the role of the HIF-1? signaling pathway in cutaneous squamous cell carcinoma (SCC). We investigated the expression of HIF-1?, vascular endothelial growth factor (VEGF) and the HIF negative regulator, prolyl hydroxylase domain protein 2 (PHD2), in cutaneous SCC, Bowens disease, seborrheic keratosis (SK) and normal skin by immunohistochemistry and in situ hybridization. Additionally, we explored the relationships between these factors and the clinical and histological characteristics of each disease. Our study indicated that the expression of HIF-1? and VEGF was significantly higher (P < 0.05) in cutaneous SCC than in Bowens disease, SK or normal skin. In contrast, PHD2 showed significantly higher expression in normal skin compared with SK, Bowens disease and cutaneous SCC (P < 0.05). Grade II-IV cutaneous SCC had higher expression levels of nuclear HIF-1? and cytoplasm VEGF protein but less nuclear PHD2 protein than grade ? cutaneous SCC (P < 0.05). Overexpression of HIF-1? and VEGF, as well as the decreased expression of PHD2, may play important roles in the development of cutaneous SCC.
Demineralized dentin matrix (DDM) had been successfully used in clinics as bone repair biomaterial for many years. However, particle morphology of DDM limited it further applications. In this study, DDM and collagen were prepared to DDM composite collagen material. The surface morphology of the material was studied by scanning electron microscope (SEM). MC3T3-E1 cells responses in vitro and tissue responses in vivo by implantation of DDM composite collagen material in bone defect of rabbits were also investigated. SEM analysis showed that DDM composite collagen material evenly distributed and formed a porous scaffold. Cell culture and animal models results indicated that DDM composite collagen material was biocompatible and could support cell proliferation and differentiation. Histological evaluation showed that DDM composite collagen material exhibited good biocompatibility, biodegradability and osteoconductivity with host bone in vivo. The results suggested that DDM composite collagen material might have a significant clinical advantage and potential to be applied in bone and orthopedic surgery.
Mononuclear cell (MNC) infiltrate is one of the earliest pathological changes in systemic sclerosis (SSc) skin. However, little is known about the recruitment of these cells into skin lesions. Recently, the role of chemokines has been suggested in the pathogenesis of SSc. Here we studied the expressions and distributions of CC chemokine CCL20 and its receptor CCR6 in early SSc skin lesions and the difference in CCL20 expressions and ability to recruite MNCs of normal dermal fibroblast (NDF) and scleroderma dermal fibroblast (SSDF). We found that the expressions of CCL20 and its receptor CCR6 were obviously up-regulated in SSc in contrast to normal human skin. mRNA levels were significantly expressed in SSc lesional skins vs normal skin tissues. SSDF displayed increased constitutive expressions of CCL20 mRNA and protein. In addition, Th1 cytokines (TNF-? and IL-1?) remarkably increased the expression of CCL20 in both NDF and SSDF in a dose- and time-dependent manner. Supernatants from SSDF showed stronger chemotactic activity to PBMCs than those from NDF. Thus our findings suggest that CCL20 released from cytokine-activated SSDF plays an important role in the induction of SSc by further recruiting more MNCs to the skin.
Chronic hepatitis B virus (HBV) infection has been strongly associated with hepatocellular carcinoma. HBV encodes an oncogenic hepatitis B virus X protein (HBx), which is a multifunctional regulator that modulates signal transduction, transcription, cell cycle progress, protein degradation, apoptosis, and genetic stability through direct and indirect interaction with host factors. The subcellular localization of HBx is primarily cytoplasmic, with a small fraction in the nucleus. In addition, high levels of HBx expression lead to an abnormal mitochondrial distribution. The dynamic distribution of HBx could be important to the multiple functions of HBx at different stages of the HBV life cycle. This short review presents an overview of the differential roles of HBx as a function of its intracellular localization.
Condyloma acuminatum (CA) is a benign epithelial tumor caused by infection with human papillomaviruses (HPVs) and characterized by abnormal cell proliferation. Cellular caspase-8 (FLICE)-like inhibitory protein (c-FLIP) was originally identified as an inhibitor of death-receptor signaling through competition with caspase-8 for recruitment to FAS-associated via death domain (FADD). More recently, it has been determined that c-FLIP is associated with the survival and proliferation of T cells and keratinocytes. The aim of this work was to study the expression of c-FLIP in CA and its relationship with keratinocyte proliferation. Immunoperoxidase staining methods were applied to analyze the location and expressions of both c-FLIP and proliferating cell nuclear antigens (PCNA) in 34 CA and 16 normal foreskin tissues. Semiquantitative reverse transcriptase-polymerase chain reactions (RT-PCR) and western blotting were performed to further identify the expression of c-FLIP in CA. c-FLIP expression at both mRNA and protein level was significantly higher in CA than normal foreskin. c-FLIP expression was highly correlated with the PCNA labeling index (LI) in CA. We concluded that c-FLIP overexpression might take part in keratinocyte proliferation in CA.
Transferrin receptor (TfR) has been used as a target for antibody-based therapy of cancer. Anti-TfR antibody together with chemotherapeutic drugs has potential for cancer therapy. In this study, we investigated the in vitro anti-tumor effects of the anti-TfR monoclonal antibody (mAb), 7579, alone or in combination with Nimustine, a chemotherapeutic drug, on the gliomas cell lines U251 and U87MG. Our results indicated that 7579 alone dramatically down-regulated surface expression of TfR on tumor cells and induced S phase accumulation and apoptosis of tumor cells. Compared with 7579 or Nimustine used alone, the combination of 7579 with Nimustine demonstrated enhanced growth inhibitory effect on tumor cells. PI (Propidium iodide)/Annexin V staining analyzed by FCM (flow cytometry) demonstrated that 7579 enhanced the cytotoxic effects of chemotherapeutic drug on tumor cells, indicating the therapeutic effect of 7579 was mediated mainly by promoting tumor cell necrosis. Using the median-effect/combination-index isobologram method, we further evaluated the nature of 7579/chemotherapeutic drug interactions. Synergistic interaction was observed for combination of 7579 with Nimustine. Our study provides additional evidence to develop combination therapies of anti-TfR mAbs-plus chemoimmunotherapy for gliomas.
HBV replicates noncytopathically in hepatocytes, but HBV or proteins encoded by HBV genome could induce cytokines, chemokines expression by hepatocytes. Moreover, liver damage in patients with HBV infection is immune-mediated and cytokines play important roles in immune-mediated liver damage after HBV infection. Interleukin-32 (IL-32) is a proinflammatory cytokine and plays a critical role in inflammation. However, the role of HBV in IL-32 expression remains unclear. In the present study, we demonstrate that hepatitis B virus protein X (HBx) increases IL-32 expression through the promoter of IL-32 at positions from -746 to +25 and in a dose-dependent manner. Furthermore, we demonstrate that increase of NF-?B subunits p65 and p50 in Huh7 cells also augments IL-32 expression, and the NF-?B inhibitor blocks the effect of HBx on IL-32 induction. These results indicate that NF-?B activation is required for HBx-induced IL-32 expression. In conclusion, IL-32 is induced by HBx in Huh7 cells. Our results suggest that IL-32 might play an important role in inflammatory response after HBV infection.
Both DNA and RNA can serve as powerful building blocks for bottom-up fabrication of nanostructures. A pioneering concept proposed by Ned Seeman 30 years ago has led to an explosion of knowledge in DNA nanotechnology. RNA can be manipulated with simplicity characteristic of DNA, while possessing noncanonical base-pairing, versatile function, and catalytic activity similar to proteins. However, standing in awe of the sensitivity of RNA to RNase degradation has made many scientists flinch away from RNA nanotechnology. Here we report the construction of stable RNA nanoparticles resistant to RNase digestion. The 2-F (2-fluoro) RNA retained its property for correct folding in dimer formation, appropriate structure in procapsid binding, and biological activity in gearing the phi29 nanomotor to package viral DNA and producing infectious viral particles. Our results demonstrate that it is practical to produce RNase-resistant, biologically active, and stable RNA for application in nanotechnology.
The expression of TfR/CD71 in T-cell surface plays a pivotal role in T-cell activation and proliferation. Anti-human-TfR monoclonal antibody could be used as an immunosuppressant in transplant therapy because of their potential to suppress T-cell responses to alloantigens. We therefore examined the feasibility of an anti-human-TfR chimeric antibody (D2C) in suppression of T-cell activation in vitro and graft-versus-host reaction (GVHR) in animals. D2C is a chimeric antibody produced by introducing the human Fc fragment. This antibody showed low antigenicity but high suppressive effect manifested by high potency to block the activation and proliferation of lymphocytes in response to alloantigens. D2C also showed capability to mediate complement-dependent cytotoxicity, which could be correlated with TfR expression in peripheral blood mononuclear cells (PBMCs). Importantly, administration of D2C significantly prolonged survival time of nude mice transplanted with human PBMCs when compared with that of control IgG-treated animals (61.2 ± 4.46 vs. 22.1 ± 5.5 days), which is associated with inhibited GVHR characterized by decreased interleukin-1 and tumor necrosis factor ? production derived from transplanted PBMCs. Human-TfR chimeric antibody such as D2C could be a valuable option for the treatment of acute form of graft-versus-host disease.
PD-L1 have been identified as the ligand for PD-1, and shown to play a role in the regulation of immune responses. In the present study, we investigated whether overexpressing PD-L1 on islet beta cells could induce negative regulation in primary and primed allogeneic lymphocyte response. pPD-L1-EGFP or pEGFPn1 were transfected in NIT-1 cells, for establishment of pPD-L1-EGFP or pEGFPn1 stable transfectants, namely NIT-PD-L1 and NIT-EGFP. In mixed cells reaction, as compared with the controls of NIT-1 or NIT-EGFP, NIT-PD-L1-primed splenocytes showed the lowest proliferative response but severe apoptosis when restimulated with NIT-PD-L1 cells in vitro. Overexpressing PD-L1 on NIT-1 cells could downregulate IFN-gamma but upregulate IL-4 and IL-10 production by the primed lymphocytes. In addition, proliferative response of primary reactive lymphocytes stimulated with NIT-PD-L1 was lower than those lymphocytes restimulated with NIT-1 cells or NIT-EGFP cells. Our data demonstrated that PD-L1 has down-regulative effects on alloimmune responses.
A fusion protein of single chain antibody (scFv) specific for transferrin receptor (TfR, CD71) and viral peptide/HLA-A2 complex was prepared in this study to redirect cytotoxic T cells (CTLs) of viral specificity to tumor cells by attaching the ligand of T cell receptor (TCR) to tumor cells via binding of TfR scFv to TfR. The results demonstrate that the fusion protein can attach the active virus-peptide/HLA-A2 complex to HLA class I-negative, TfR-expressing K562 cells through binding of TfR scFv to TfR, and mediate cytotoxicity of viral peptide-specific CTLs against K562 cells in vitro. In addition, the fusion protein can induce inhibition of solid tumor formation and improve survival time in tumor xenograft nude mouse with the injection of the sorted viral peptide-specific CTLs generated by co-culture of peripheral blood lymphocytes from HLA-A2 positive donors with inactivated T2 cells pulsed with the viral peptide.
Alternatively activated dendritic cell (aaDC) can prolong allograft survival in the mouse model. However, the molecular mechanism(s) by which these DCs function to regulate alloreactive T-cell responses remains to be clearly defined.
Islet transplantation is considered a therapeutic option for type 1 diabetes (T1D). However, allorejection is one major barrier for the successful islet transplantation. In the present study, we have tested the feasibility of a deletion construct for Fas-associated death domain protein (FADD; without the death effecter domain) fused with green fluorescent protein (FADDdel-GFP) for blocking the Fas-FasL signaling pathway in prevention of transplanted beta cell destruction by allo-rejection in T1D. In vitro studies have shown that NIT-1 cells with ectopic FADDdel expression were resistant to cytokine-induced apoptosis and CTL-mediated lysis. Diabetic Balb/c mice reached normoglycemia promptly and gained weight after transplantation of NIT-1 cells with ectopic FADDdel-GFP expression. These recipients showed a significant longer survival time than that of recipients transplanted with NIT cells with ectopic GFP expression only. Our results together suggest that FADDdel could be a useful target for the improvement of islet transplantation for T1D.
The pRNA (packaging RNA) of bacteriophage phi29 DNA packaging motor has been reported to have novel applications in nanotechnology and nanomedicine. The unique ability of pRNA to form dimers, trimers, hexamers and patterned superstructures via the interaction of two reengineered interlocking loops makes it a promising polyvalent vehicle to load siRNA and other therapeutic molecules and be applied as a therapeutic nanoparticle in tumor therapy. In this study, several tumor cell lines were used to evaluate the previously reported pRNA nanotechnology for specific siRNA delivery and for the silencing of targeted genes. It was found that MCF-7 and HeLa cells, out of twenty-five tested tumor cell lines, expressed high levels of folate receptors and exhibited specific binding of the FITC-folate-pRNA nanoparticles, while the others expressed low levels and thus, for these, delivery was not feasible using folate as a targeting agent. Folate receptor positive tumor cells were then incubated with the chimeric pRNA dimer harboring both the folate-pRNA and the chimeric pRNA/siRNA (survivin). Knock down effects of survivin expression in these tumor cells were detected at the mRNA level by real time-PCR and at the protein level by western blot. Apoptosis was detected by flow cytometry analysis with dual staining of annexinV-FITC and PI. The data suggest that the chimeric pRNA nanoparticles containing folate-pRNA and pRNA/siRNA (survivin) could be specifically taken up by tumor cells through folate receptor-mediated endocytosis, resulting in significant inhibition of both transcription and expression of survivin in tumor cells and triggering cell apoptosis. Using such protein-free nanoparticles as therapeutic reagents would not only allow specific gene delivery and extend the in vivo retaining time but also allow long-term administration of therapeutic particles, therefore avoiding the induction of antibodies caused by repeated treatment for chronic diseases.
Programmed death ligand (PDL) is a new member of the B7 family of costimulatory molecules that specifically interacts with programmed death 1 (PD-1) expressed on activated T cells, B cells, and myeloid cells. Collagen II (CII)-induced arthritis (CIA) is an experimental model of arthritis that has been used to dissect the pathogenesis of human rheumatoid arthritis. In this study, we have investigated the effects of PDL-Ig on CIA. Administration of PDL-Ig significantly ameliorated the disease as assessed by clinical arthritis score and histology in the joints. Expression of proinflammatory cytokines, such as IL-17 and IL-23, in the serum was reduced by PDL-Ig treatment. These results showed a beneficial effect of PDL-Ig on CIA through anti-inflammatory actions and inhibition of cell proliferation in response to CII, suggesting that the PD-1-PDL pathway may be involved in the pathogenesis of CIA, and thus PDL-Ig may be a useful therapy for the improvement of human rheumatoid arthritis.
Generation and adoptive transfusion of alloreactive cytotoxic T lymphocytes (CTLs) specific for tumor are expected to circumvent tumor tolerance. Here we describe a novel protocol to raise allo-restricted, peptide-specific CTLs by co-culture of murine splenocytes and autologous macrophage bearing an allogeneic H-2K molecule associated with its restricted peptide (peptide/allo-MHC). The extracellular domains of H-2K(d) were fused with constant domains of murine IgG2a heavy chain to generate a fusion protein (peptide/H-2K(d)/IgG2aFc, the dimer) consisting of divalent TCR-ligands and an IgG Fc receptor type I (FcgammaRI)-reactive moiety. The dimer is able to bind to macrophage (Mvarphi) of H-2K(k) via the interaction of the Fc part with FcgammaRI, and cause the H-2K(k) Mvarphi to be coated with the peptide/H-2K(d) complex. The results show that proliferation of CD8+ cells is enhanced and that the specific-tetramer stained CD8+ cells appear more frequently by co-culture of H-2K(k) splenocytes with the autologous Mvarphi loaded with the dimer. Furthermore, the CD8+ T cells from the co-cultural bulk exhibit an elevated cytotoxicity against a specific target (H-2K(d)-restricted, peptide-specific cytotoxicity), compared with that against the irrelevant targets. This study provides a strategy for preparation of allo-restricted, peptide-specific CTLs, which may add to our arsenal for adoptive immunotherapy to eliminate chronic virally infected or tumor cells.
The graft-versus-leukemia effect of allogeneic marrow transplantation suggests the dramatic effect of the allogeneic T cell to eradicate malignant disease. Preparation and adoptive transfusion of tumor-specific T cells from HLA-mismatched donors might be expected to circumvent CTL tolerance to the tumor. In this study, a soluble, divalent HLA-A2 molecule was constructed with the Fc part of human IgG1 and was pulsed with a peptide related to melanoma tyrosinase 368-376 [Tyr(368-376) (Tyr)] to form the Tyr/HLA-A2 dimer, which allowed loading onto monocytes via interaction of the Fc and FcR. The HLA-A2-negative (HLA-A2-ve) monocytes loaded with the Tyr/HLA-A2 dimer acted as allo-APC with copies of a single allogeneic epitope. After coculture of the HLA-A2-ve PBLs and autologous monocytes loaded with the dimer, CD8+ cells in the coculture show an obvious proliferation and increased frequency of Tyr/HLA-A2 tetramer-stained cells. The sorted Tyr/HLA-A2 tetramer-positive CD8+ cells display an elevated cytotoxic activity against HLA-A2-positive melanoma cells expressing tyrosinase endogenously (i.e., SK-Mel-5) but little against tyrosinase-negative melanoma cells (i.e., A375). The coculture of PBLs and autologous monocytes loaded with allogeneic peptide/HLA complexes offers a novel approach to expand allo-restricted, peptide-specific CTLs, which might be a potential arsenal for treatment of patients with malignant disease, if the tumor-related epitope were defined.
Malignant melanoma (MM) is a major public health problem. The development of effective, systemic therapies for MM is highly desired. We showed here that the transferrin receptor (TfR) was a suitable surface marker for targeting of gene therapy in MM and that the hypoxia-inducible factor-1alpha (HIF-1alpha) was an attractive therapeutic molecular target in MM. We observed that inhibition of HIF-1alpha blocked cell proliferation and induced cell apoptosis in vitro. We then showed that a transferrin-polyethylenimine-HIF-1alpha-short-hairpin RNA (Tf-PEI-HIF-1alpha-shRNA) complex could target MM specifically and efficiently both in vivo and in vitro, exploiting the high expression of the TfR in MM. The systemic delivery of sequence-specific small-interfering RNA (siRNA) against HIF-1alpha by the Tf- PEI-HIF-1alpha-shRNA complex dramatically inhibited tumor growth in the A375 MM xenograft model. The underlying concept of transfecting a HIF-1alpha shRNA expression vector complexed with Tf-PEI to block HIF-1alpha holds promise as a clinical approach to gene therapy for MM.
Human leukocyte antigen (HLA)-G, a nonclassical HLA class I molecule, induces a wide range of tolerogenic immunological effects by means of interaction with its inhibitory receptors. However, recent studies show that HLA-G dimer formation is essential to bind to its receptors and exhibit its effects.
Herpes simplex virus thymidine kinase (HSV-TK) gene and dendritic cells (DC) have been used as the pioneering in cancer therapy. HSV-TK gene can induce apoptosis and necrosis in tumor cells in the presence of the non-toxic prodrug ganciclovir (GCV). We investigated the anti-tumor effect of DC vaccination by introducing dying cells from HSV-TK gene treatment as an adjuvant. HepG(2)-TK cell line was established by transfecting human hepatoma cell line HepG(2) (HLA-A(2) positive) with HSV-TK gene. Dying tumor cells were generated by culturing HepG(2)-TK cells with GCV. After engulfed dying cells efficiently, immature DCs (imDC) derived from human monocytes were fully matured and elicited marked proliferation and cytotoxicity against HLA matched HepG(2) cells in autologous peripheral blood mononuclear cells (PBMC). It also implied that HepG(2) specific CTLs played an important role in the cytotoxicity which was primarily depended on Th1 responses. Given the feasibility of inducing dying cells by HSV-TK/GCV in vivo, our results suggest an effective method in clinical human hepatocellular carcinoma (HCC) treatment by an in vitro model of applying HSV-TK gene modified human tumor cells integrated with DC vaccination.
Raising tumor-specific allorestricted T cells in vitro for adoptive transfusion is expected to circumvent host tumor tolerance. However, it has been assumed that alloreactive T cell clones activated in vitro ranges from peptide-specific with high avidity to peptide-degenerate with low avidity. In this study, we examined the peptide specificity and cross-reactivity of T cell responses in vitro to an allogeneic epitope and a nominal epitope with a modified co-culture of lymphocytes and autologous monocytes. After binding to the monocyte via the interaction of its Fc part and the cell surface IgG Fc receptor type I (Fc?RI), a fusion protein consisting of the extracellular domains of HLA-A2 molecule and the Fc region of IgG1 (the dimer) introduced a single epitope into the co-culture. The dimer-coated monocytes stimulated the proliferation of autologous CD8(+) T cells after co-culturing. The CD8(+) T cell responses were self-HLA-restricted for HLA-A2-positive (HLA-A2+ve) samples and allo-HLA-restricted for HLA-A2-negative (HLA-A2-ve) samples, since the co-cultural bulks stained with HLA-A2 tetramers, human interferon-gamma (IFN-?) production in response to T cell receptor (TCR) ligands, and cytotoxicity against a panel of target cells exhibited peptide-specific properties. Two HLA-A2-restricted peptides with sequence homology were included, allowing the comparison of cross-reactivity between allo-antigen- and nominal antigen-induced CD8(+) T cell responses. Interestingly, the allo- and self-HLA-restricted CD8(+) T cell responses were similar in the peptide cross-reactivity, although the allorestricted T cell response seemed, overall, more intensive and had higher binding affinity to specific tetramer. Our findings indicated the alloreactive T cells raised by the co-culture in vitro were as peptide specific and cross-reactive as the self-HLA-restricted ones.
The development of vectors for cell-specific gene delivery is a major goal of gene therapeutic strategies. Transferrin receptor (TfR) is an endocytic receptor and identified as tumor relative specific due to its overexpression on most tumor cells or tissues, and TfR binds and intakes of transferrin-iron complex. We have previously generated an anti-TfR single-chain variable fragments of immunoglobulin (scFv) which were cloned from hybridoma cell line producing antibody against TfR linked with a 20 aa-long linker sequence (G4S)4. In the present study, the anti-TfR single-chain antibody (TfRscFv) was fused to DNA-binding domain of the yeast transcription factor GAL4. The recombinant fusion protein, designated as TfRscFv-GAL4, is expected to mediate the entry of DNA-protein complex into targeted tumor cells.
This study evaluated the performance of the Maxwell 16 System (Promega) for extraction of influenza virus (flu-v) RNA from diverse samples compared to a classical manual method (QIAamp Kit, QIAGEN). Following extraction by the two methods, all samples were analyzed by Real-time RT-PCR. Results revealed that the use of the standard Maxwell 16 protocol (Maxwell 16-S) resulted in good linearity and precision across a wide concentration range and higher sensitivity of detection from flu-v stock suspensions than the manual method. Compared with the latter method, Maxwell 16-S extracted RNA more efficiently (higher RNA yield and/or fewer PCR inhibitors) from throat swabs and bronchoalveolar lavage fluids, while both methods performed comparably on fecal samples from human and poultry in terms of overall threshold cycle values and detection rates although the Maxwell 16-S co-purified more inhibitors from fecal samples. The capacity of this system to remove inhibitors from fecal matrix was improved by using a modified Maxwell 16 protocol with a reduced sample input, which eliminated all false-negatives produced by the Maxwell 16-S. These findings suggest that the Maxwell 16 System is suitable for RNA extraction from multiple-source samples for diagnosis of influenza and viral load determination and that a proper reduction in starting sample volume may improve the detection of flu-v from complex matrices such as feces. Additionally, this system allows flexible sample throughput and labor-saving sample processing with little or no risk of cross-contamination.
Hepatitis B virus (HBV) infection is a worldwide liver disease and nearly 25% of chronic HBV infections terminate in hepatocellular carcinoma (HCC). Currently, there is no effective therapy to inhibit HBV replication and to eliminate hepatoma cells, making it highly desired to develop novel therapies for these two stages of the HBV-caused detrimental disease. Recently, short hairpin RNA (shRNA) has emerged as a potential therapy for virus-infected disease and cancer. Here, we have generated a shRNA, pGenesil-siHBV4, which effectively inhibits HBV replication in the human hepatoma cell line HepG2.2.15. The inhibitory effects of pGenesil-siHBV4 are manifested by the decrease of both the HBV mRNA level and the protein levels of the secreted HBV surface antigen (HBsAg) and HBV e antigen (HBeAg), and by the reduction of secreted HBV DNA. Using mouse hydrodynamic tail vein injection, we demonstrate that pGenesil-siHBV4 is effective in inhibiting HBV replication in vivo. Because survivin plays a key role in cancer cell escape from apoptosis, we further generated pGenesil-siSurvivin, a survivin-silencing shRNA, and showed its effect of triggering apoptosis of HBV-containing hepatoma cells. To develop targeted shRNA therapy, we have identified that as a specific binder of the asialoglycoprotein receptor (ASGPR), jetPEI-Hepatocyte delivers pGenesil-siHBV4 and pGenesil-siSurvivin specifically to hepatocytes, not other types of cells. Finally, co-transfection of pGenesil-siHBV4 and pGenesil-siSurvivin exerts synergistic effects in inducing hepatoma cell apoptosis, a novel approach to eliminate hepatoma by downregulating survivin via multiple mechanisms. The application of these novel shRNAs with the jetPEI-Hepatocyte targeting strategy demonstrates the proof-of-principle for a promising approach to inhibit HBV replication and eliminate hepatoma cells with high specificity.
The MOG35-55 peptide-induced experimental autoimmune encephalomyelitis (EAE) model in C57BL/6 mice is a useful animal model to explore therapeutic approaches to T cell-mediated autoimmune diseases because the dominant T-cell epitope(s) have been defined. It is rational that antigen-specific immunosuppression can be induced by using MHC-peptide complexes as specific TCR ligand(s) that interact with autoreactive T cells in the absence of co-stimulation. In this study, a soluble divalent MOG35-55/I-A(b) fusion protein (MOG35-55/I-A(b) dimer) was constructed to specifically target the autoreactive CD4+ T cells in the EAE mouse. Intraperitoneal administration of the MOG35-55/I-A(b) dimer significantly delayed and ameliorated EAE symptoms by reducing EAE-related inflammation in the mouse CNS and reducing encephalitogenic Th1 and Th17 cells in the peripheral lymphoid organs. We observed that dimer intervention at a concentration of 1.2 nM suppressed MOG35-55 peptide-specific 2D2 transgenic T cells (2D2 T cells) proliferation by over 90% after in vitro activation with MOG35-55 peptide. The mechanisms involved in this antigen-specific dimer-mediated suppression were found to be downregulated TCR-CD3 expression as well as upregulated expression of membrane-bound TGF-? (mTGF-?) and IL-10 suppressive cytokines by the autoreactive CD4+ T cells. Collectively, our data demonstrates that soluble divalent MHC class II molecules can abrogate pathogenic T cells in EAE. Furthermore, our data suggests that this strategy may provide an efficient and clinically useful option to treat autoimmune diseases.
Transferrin receptor (TfR) has been used as a target for the antibody-based therapy of cancer due to its higher expression in tumors relative to normal tissues. Great potential has been shown by anti-TfR antibodies combined with chemotherapeutic drugs as a possible cancer therapeutic strategy. In our study, we investigated the anti-tumor effects of anti-TfR monoclonal antibody (mAb) alone or in combination with sinomenine hydrochloride in vitro. Results suggested that anti-TfR mAb or sinomenine hydrochloride could induce apoptosis, inhibit proliferation, and affect the cell cycle. A synergistic effect was found in relation to tumor growth inhibition and the induction of apoptosis when anti-TfR mAb and sinomenine hydrochloride were used simultaneously. The expression of COX-2 and VEGF protein in HepG2 cells treated with anti-TfR mAb alone was increased in line with increasing dosage of the agent. In contrast, COX-2 expression was dramatically decreased in HepG2 cells treated with sinomenine hydrochloride alone. Furthermore, we demonstrated that the inhibitory effects of sinomenine hydrochloride and anti-TfR mAb administered in combination were more prominent than when the agents were administered singly. To sum up, these results showed that the combined use of sinomenine hydrochloride and anti-TfR mAb may exert synergistic inhibitory effects on human hepatoma HepG2 cells in a COX-2-dependent manner. This finding provides new insight into how tumor cells overcome the interference of iron intake to survive and forms the basis of a new therapeutic strategy involving the development of anti-TfR mAb combined with sinomenine hydrochloride for liver cancer.
The role of interleukin-32 (IL-32) in chronic hepatitis B (CHB) remains unclear. In order to identify the role of IL-32 in CHB, we detected the expression levels of IL-32 in liver samples of CHB patients and analyzed the correlation between IL-32 and liver inflammation/fibrosis.
Birc5 (previously known as survivin) is a cancer-specific protein. Due to the upregulation of its expression in various human malignancies and its key role in apoptosis, proliferation and angiogenesis, Birc5 has attracted attention as a target for anticancer therapies. In this study, when Birc5 was silenced in HepG2 cells, 29.7±3.3% cells underwent apoptosis as expected. It was found that the expression levels of glucose-regulated protein 78 (Hspa5, previously known as Grp78) was increased by almost 3-fold in Birc5-silenced HepG2 cells. Hspa5, a master regulator of the anti-apoptotic unfolded protein response signalling network, can also promote tumor proliferation, survival and metastasis. Hence, we hypothesized that the co-silencing of Birc5 and Hspa5 may exert a stronger apoptosis-inducing effect than single gene interference. To verify this, the expression levels of Birc5 and Hspa5 in human hepatocellular carcinoma tissues were determined. Immunohistochemical staining showed that the expression of Birc5 and Hspa5 was elevated in 28 out of 31 samples. Additionally, plasmid-based siRNA against Birc5 and/or Hspa5 were constructed and transfected into the human hepatocellular liver carcinoma cell line, HepG2. Compared with the HepG2 cells, in which Birc5 or Hspa5 were silenced alone, only 44.2±3.4% of the co-silenced cells proliferated, and 40.3±3.7% co-silenced cells underwent apoptosis (p<0.05). Furthermore, tumor formation from inoculated subcutaneous co-silenced cells in nude mice was inhibited significantly. The current study suggests that Birc5 and Hspa5 could be important survival factors for hepatoma carcinoma cells and that the simultaneous knockdown of Birc5 and Hspa5 is more effective in inducing apoptosis in HepG2 cells than the knockdown of Birc5 or Hspa5 alone. The co-silencing of Birc5 and Hspa5 could be warranted for cancer therapy.
Generation of tumor specific alloreactive CD4(+) T cells is important to circumvent tumor tolerance. Here, we generate allorestricted peptide-specific CD4(+) T cells by coculture of lymphocytes and autologous monocytes bearing allogeneic HLA-DR15 molecule associated with its restricted peptide. Binding of a dimeric HLA-DR15/IgG1-Fc fusion protein (the dimer) to HLA-DR15 negative (HLA-DR15-ve) monocytes made the monocytes coated with the allogeneic epitope. An increased proliferation of CD4(+) T cells and induction of Th1 cells appeared after coculturing of HLA-DR15-ve lymphocytes and the autologous monocytes loaded with the dimer. The cocultural bulks showed an increased frequency of the specific dimer-stained CD4(+) T cells and the expanded CD4(+) T cells exhibited an elevated IFN-? production in response to specific TCR ligand. Tumor rejection effects of the allorestricted E7-specific CD4(+) T cells raised by the coculture were observed in nude mice challenged with human cervical cancer cell SiHa expressing both HLA-DR15 and E7 antigens, as the tumor avoidance and life span of the mice were improved after adoptive transfer of the CD4(+) T cells. This study may help to develop strategies to separate graft-versus-leukemia or graft-versus-tumor reaction from graft-versus-host disease, and add to the pool of human high-avidity TCRs specific for tumor or virus antigens.
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