Actions of Parathyroid Hormone Ligand Analogues in Humanized PTH1R Knockin Mice.

Rodent models are commonly used to evaluate parathyroid hormone (PTH) and PTH-related protein (PTHrP) ligands and analogues for their pharmacologic activities and potential therapeutic utility toward diseases of bone and mineral ion metabolism. Divergence, however, in the amino acid sequences of rodent and human PTH receptors (rat and mouse PTH1Rs are 91% identical to the human PTH1R) can lead to differences in receptor-binding and signaling potencies for such ligands when assessed on rodent vs human PTH1Rs, as shown by cell-based assays in vitro. This introduces an element of uncertainty in the accuracy of rodent models for performing such preclinical evaluations. To overcome this potential uncertainty, we used a homologous recombination-based knockin (KI) approach to generate a mouse (in-host strain C57Bl/6N) in which complementary DNA encoding the human PTH1R replaces a segment (exon 4) of the murine PTH1R gene so that the human and not the mouse PTH1R protein is expressed. Expression is directed by the endogenous mouse promoter and hence occurs in all biologically relevant cells and tissues and at appropriate levels. The resulting homozygous hPTH1R-KI (humanized) mice were healthy over at least 10 generations and showed functional responses to injected PTH analog peptides that are consistent with a fully functional human PTH1R in target bone and kidney cells. The initial evaluation of these mice and their potential utility for predicting behavior of PTH analogues in humans is reported here.

Chryseobacterium vaccae sp. nov., isolated from raw cow's milk.

Strain CA7T, a Gram-stain-negative, non-motile, non-spore-forming, aerobic and rod-shaped bacterial strain, was isolated from raw cow's milk collected from a farm affiliated with Chung-Ang University, Anseong, Korea, and characterized by a polyphasic approach. Optimal growth of strain CA7T was observed on tryptic soy agar at 30 °C and pH 7.0 with 0 % NaCl. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain CA7T belonged to the genus Chryseobacterium. The most closely related strains (16S rRNA gene sequence similarity indicated in parentheses), based on the phylogenetic analysis, were Chryseobacterium rhizosphaerae KCTC 22548T (98.08 %), Chryseobacterium nakagawai CCUG 60563T (98.61 %), Chryseobacterium jejuense KACC 12501T (97.85 %) and Chryseobacterium aurantiacum KCTC 62135T (97.78 %). Whole genome sequencing indicated that the genome size was 5 125 723 bp and had a DNA G+C content of 37.4 mol%. Average nucleotide identity values for strain CA7T with C. rhizosphaerae, C. nakagawai, C. jejuense, C. aurantiacum, and the type species of the genus Chryseobacterium, C. gleum, were 80.2, 79.8, 79.8, 79.6 and 80.4 %, respectively. The digital DNA-DNA hybridization values of CA7T compared to C. rhizosphaerae, C. nakagawai, C. jejuense, C. aurantiacum and C. gleum were 24.1, 23.9, 23.9, 23.7 and 24.3 %, respectively. The major fatty acids were iso-C15 : 0, summed feature 9 (iso-C17 : 1 ω9c and/or C16 : 0 10-methyl), iso-C17 : 0 3-OH and summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1 ω7c). Menaquinone-6 was the only respiratory quinone. The major polar lipid was phosphatidylethanolamine. Based on this polyphasic taxonomic study, strain CA7T represents a novel species of the genus Chryseobacterium for which the name Chryseobacterium vaccae sp. nov. is proposed. The type strain is CA7T (=KACC 21402T=JCM 33749T).

Acinetobacter pullorum sp. nov., Isolated from Chicken Meat.

A bacterial strain, designated B301T and isolated from raw chicken meat obtained from a local market in Korea, was characterized and identified using a polyphasic taxonomic approach. Cells were gram-negative, non-motile, obligate-aerobic coccobacilli that were catalase-positive and oxidase-negative. The optimum growth conditions were 30°C, pH 7.0, and 0% NaCl in tryptic soy broth. Colonies were round, convex, smooth, and cream-colored on tryptic soy agar. Strain B301T has a genome size of 3,102,684 bp, with 2,840 protein-coding genes and 102 RNA genes. The 16S rRNA gene analysis revealed that strain B301T belongs to the genus Acinetobacter and shares highest sequence similarity (97.12%) with A. celticus ANC 4603T and A. sichuanensis WCHAc060041T. The average nucleotide identity and digital DNA-DNA hybridization values for closely related species were below the cutoff values for species delineation (95-96% and 70%, respectively). The DNA G+C content of strain B301T was 37.0%. The major respiratory quinone was Q-9, and the cellular fatty acids were primarily summed feature 3 (C16:1 ω6c/C16:1 ω7c), C16:0, and C18:1 ω9c. The major polar lipids were phosphatidylethanolamine, diphosphatidyl-glycerol, phosphatidylglycerol, and phosphatidyl-serine. The antimicrobial resistance profile of strain B301T revealed the absence of antibiotic-resistance genes. Susceptibility to a wide range of antimicrobials, including imipenem, minocycline, ampicillin, and tetracycline, was also observed. The results of the phenotypic, chemotaxonomic, and phylogenetic analyses indicate that strain B301T represents a novel species of the genus Acinetobacter, for which the name Acinetobacter pullorum sp. nov. is proposed. The type strain is B301T (=KACC 21653T = JCM 33942T).

Positive effects of bisphosphonates on bone and muscle in a mouse model of Duchenne muscular dystrophy.

Patients with Duchenne muscular dystrophy are at increased risk of decreased bone mineral density and bone fracture as a result of inactivity. To determine if antiresorptive bisphosphonates could improve bone quality and their effects on muscle we studied the Mdx mouse, treated with pamidronate during peak bone growth at 5 and 6 weeks of age, and examined the outcome at 13 weeks of age. Pamidronate increased cortical bone architecture and strength in femurs with increased resistance to fracture. While overall long bone growth was not affected by pamidronate, there was significant inhibition of remodeling in metaphyseal trabecular bone with evidence of residual calcified cartilage. Pamidronate treatment had positive effects on skeletal muscle in the Mdx mice with decreased serum and muscle creatine kinase and evidence of improved muscle histology and grip strength.

Prostaglandin receptor EP4 in abdominal aortic aneurysms.

Abdominal aortic aneurysm (AAA) pathogenesis is distinguished by vessel wall inflammation. Cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase-1, key components of the most well-characterized inflammatory prostaglandin pathway, contribute to AAA development in the 28-day angiotensin II infusion model in mice. In this study, we used this model to examine the role of the prostaglandin E receptor subtype 4 (EP4) and genetic knockdown of COX-2 expression (70% to 90%) in AAA pathogenesis. The administration of the prostaglandin receptor EP4 antagonist AE3-208 (10 mg/kg per day) to apolipoprotein E (apoE)-deficient mice led to active drug plasma concentrations and reduced AAA incidence and severity compared with control apoE-deficient mice (P < 0.01), whereas COX-2 genetic knockdown/apoE-deficient mice displayed only a minor, nonsignificant decrease in incidence of AAA. EP4 receptor protein was present in human and mouse AAA, as observed by using Western blot analysis. Aortas from AE3-208-treated mice displayed evidence of a reduced inflammatory phenotype compared with controls. Atherosclerotic lesion size at the aortic root was similar between all groups. In conclusion, the prostaglandin E(2)-EP4 signaling pathway plays a role in the AAA inflammatory process. Blocking the EP4 receptor pharmacologically reduces both the incidence and severity of AAA in the angiotensin II mouse model, potentially via attenuation of cytokine/chemokine synthesis and the reduction of matrix metalloproteinase activities.

Transfer of Her-2/neu specificity into cytokine-induced killer (CIK) cells with RNA encoding chimeric immune receptor (CIR).

BACKGROUND: Efficient RNA transfer to dendritic cell and T cells by electroporation have been successfully applied for immunotherapy. Herein, RNA electroporation was used to transfer antigen-specific receptor (scFv) gene to cytokine-induced killer cells (CIK). METHODS: CIK was generated from peripheral blood mononuclear cells with anti-CD3 antibody, interleukin-2, and interferon (IFN)-gamma for 14 days and showed typical characteristics of CIK expressing both CD3+ and CD56+ markers and NKG2D+. CIK could lyse K562 cells, but not SKOV3 and MCF7/Her-2/neu. RESULTS: After RNA encoding anti-Her-2/neu chimeric immune receptor (CIR) with signaling portion of CD28 and CD3zeta was electroporated to CIK, more than 95% of CIK expressed anti-Her-2/neu CIR (CIR-CIK). CIR-CIK was able to produce cytokines including IFN-gamma, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha, and show cytotoxicity specific to tumor cell lines expressing Her-2/neu, SKOV3, and MCF7/Her-2/neu. Adoptive transfer of CIR-CIK in SKOV3 xenograft nude mice model led to significant inhibition of tumor growth compared with transfer of mock-transduced CIK and showed higher inhibition than that of Herceptin, humanized monoclonal antibody specific for Her-2/neu. These results suggest that RNA transfer is the convenient and efficient strategy to introduce antigen-specificity into CIK and provide potential therapeutic value of CIR-CIK in the treatment of tumors.

Selective addition of CXCR3(+) CCR4(-) CD4(+) Th1 cells enhances generation of cytotoxic T cells by dendritic cells in vitro.

Increasing importance is being given to the stimulation of Th1 response in cancer immunotherapy because its presence can shift the direction of adaptive immune responses toward protective immunity. Based on chemokine receptor expression, CXCR3(+) CCR4(-) CD4(+) T cells as Th1-type cells were investigated its capacity in monocyte-derived dendritic cell (DC) maturation and polarization, and induction of antigen specific cytotoxic T lymphocytes (CTL) in vitro. The levels of IL-4, IL-5 and IL-10 were decreased to the basal level compared with high production of IFN-gamma, TNF-alpha, and IL-2 in CXCR3+CCR4-CD4+ T cells stimulated with anti-CD3 and anti-CD28 antibodies. Co-incubation of activated CD4(+) or CXCR3(+) CCR4-CD4(+) T cells with DC (CD4(+/) DC or CXCR3(+) CD4(+/) DC, respectively) particularly up-regulated IL-12 and CD80 expression compared with DC matured with TNF-a and LPS (mDC). Although there was no significant difference between the effects of the CXCR3(+) CCR4(-) CD4(+) and CD4(+) T cells on DC phenotype expression, CXCR3(+) CD4(+/) DC in CTL culture were able to expand number of CD8(+) T cells and increased frequencies of IFN-gamma secreting cells and overall cytolytic activity against tumor antigen WT-1. These results demonstrated that the selective addition of CXCR3(+) CCR4(-) CD4(+) T cells to CTL cultures could enhance the induction of CTLs by DC in vitro, and implicated on a novel strategy for adoptive T cell therapy.

Efficient generation of survivin-specific cytotoxic T lymphocytes from healthy persons in vitro: quantitative and qualitative effects of CD4+ T cells.

For the adoptive immunotherapy and the study of cytotoxic T lymphocytes (CTLs) in human, efficient in vitro generation of CTLs is needed. However, it is still difficult to induce T cells specific for naïve antigens in vitro even though dendritic cells (DCs) as potent APCs are used. In this study, we investigated quantitative and qualitative effects of CD4+ T cells during in vitro stimulation of CD8+ T cells from healthy donors using DCs transduced with adenovirus vector expressing human survivin (Adv-survivin). CTLs were not efficiently induced in the absence of CD4+ T cells or in CD25+ depleted CD4+ T cells. When the ratio of CD4+:CD8+ T cells was quantitatively decreased from 2:1 to 1:2, proliferation of CTLs specific for survivin was gradually increased. Because DCs pulsed with HCMV pp65 protein could activate CD4+ T cells to secrete Th1 cytokines, the use of pp65 protein as an adjuvant induced higher numbers and frequencies of CTLs. Furthermore, Th1 conditioning of CD4+ T cells augmented this generation of CTLs. These results suggest that both quantitative and qualitative modulation of CD4+ T cells including the number and Th1 polarization may be required for the efficient induction of CTLs specific for tumor antigens in vitro.

A membrane-bound form of IL-4 enhances proliferation and antigen presentation of CD40-activated human B cells.

CD40-activated B (CD40-B) cells might be an attractive source of autologous antigen-presenting cells (APCs) for immunotherapy due to the ability to obtain them from peripheral blood and expand them in vitro. However, soluble IL-4 (sIL-4) in B-cell culture may not represent the "immunological synapse" between B and CD4+ T cells. In this study, the K562 cell line, which expresses CD40L and membrane-bound IL-4 (mbIL-4), could induce higher B-cell proliferation and antigen-presenting surface molecules, including adhesion, costimulatory and HLA molecules, compared with sIL-4. The differentiation to plasmablasts was decreased in CD40-B cells treated with mbIL-4 (CD40-B/mbIL-4) based on flow cytometry analysis. Furthermore, CD40-B/mbIL-4 cells were as potent as mature dendritic cells in the allogeneic lymphocyte reaction and the ability to generate cytotoxic T lymphocytes specific for cytomegalovirus pp65 antigen in vitro. Our results suggest that mbIL-4 could be used to generate CD40-B cells as potent APCs for cellular vaccines and adoptive immunotherapy.

Adoptive transfer of Epstein-Barr virus-specific cytotoxic T-lymphocytes for the treatment of angiocentric lymphomas.

Angiocentric lymphoma, known as natural killer (NK)/T-cell non-Hodgkin's lymphoma, has been reported to be associated with the Epstein-Barr virus (EBV). We performed adoptive transfer of EBV-specific polyclonal T-cell lines in 3 patients with extranodal NK/T-cell lymphoma, nasal type, and evaluated the treatment for safety, immunologic reconstitution, and clinical outcomes. The tissue samples collected from the 3 patients were confirmed by polymerase chain reaction analysis to be EBV positive. In the cases of the first and second patients, EBV-transformed B-lymphoblastoid cell lines (LCLs) and T-cell lines were generated from peripheral lymphocytes of HLA-matched sibling donors. The third patient's T-cell lines were induced with autologous lymphocytes. Polyclonal T-cell infusion was carried out after high-dose radiotherapy because active relapsed disease remained in all of the patients. The first patient received 4 weekly infusions of 2 3 10(7) cells/m(2), and the second and third patients underwent treatment with 2 cycles of infusions of the same dosage. All T-cell lines showed >60% NK activity, cytotoxic T-lymphocyte (CTL) responses of >40% against autologous LCLs, and no CTL activity against patient-derived lymphoblasts. The level of cytotoxicity increased substantially in all patients after cell infusion. The 2 patients who received T-cell therapy twice had stabilized disease for more than 3 years. These safe treatments exhibited no severe inflammatory response, and no serious toxicity developed during T-cell therapy. Our findings demonstrate that adoptively transferred cells may provide reconstitution of EBV-specific CTL responses in patients with active relapsed angiocentric lymphoma. These results provide a rationale for the immunotherapy of angiocentric lymphoma.

Activation of B cells using Schneider 2 cells expressing CD40 ligand for the enhancement of antigen presentation in vitro.

CD40 ligand (CD40L) expressed by activated CD4+ T cells is a family member of membrane bound TNF family ligand and its interaction with CD40 expressed in APC has been shown to contribute in enhancing immune response. Exogenous stimulation through CD40 has been performed using soluble trimeric CD40L, anti-CD40 monoclonal antibody and cells expressing CD40L. Schneider 2 (S2) cells, a cell line derived from Drosophila melanogaster, was transfected with a plasmid vector, pAc5.1/V5-HisA, for the constitutive expression of CD40L (S2-CD40L). Upon incubation of S2-CD40L with B-lymphocytes for 6 days, activated B cells were examined by counting B cell numbers and for activation markers including CD86 and HLA Class II molecules. The activated B cells were tested for its efficient APC function by mixed lymphocyte reactions (MLR) and enzyme-linked Immunospot (ELISPOT) assay. S2-CD40L was cultured for a year and maintained CD40L expression (>90%). S2-CD40L induced B cell activation as demonstrated by increment of total B cells and up-regulation of CD86 and MHC Class II molecules. Activated B cells pulsed with peptide from human cytomegalovirus pp65 antigen efficiently induced both proliferation and IFN-gamma secretion of T cells. Our result suggests that S2-CD40L can efficiently and conveniently generate B cells as a functional APC and represents a potential role for B-cell mediated cancer immunotherapy.