Identification and profiling of novel α1A-adrenoceptor-CXC chemokine receptor 2 heteromer

We have provided the first evidence for specific heteromerization between the α(1A)-adrenoceptor (α(1A)AR) and CXC chemokine receptor 2 (CXCR2) in live cells. α(1A)AR and CXCR2 are both expressed in areas such as the stromal smooth muscle layer of the prostate. By utilizing the G protein-coupled receptor (GPCR) heteromer identification technology on the live cell-based bioluminescence resonance energy transfer (BRET) assay platform, our studies in human embryonic kidney 293 cells have identified norepinephrine-dependent β-arrestin recruitment that was in turn dependent upon co-expression of α(1A)AR with CXCR2. These findings have been supported by co-localization observed using confocal microscopy. This norepinephrine-dependent β-arrestin recruitment was inhibited not only by the α(1)AR antagonist Terazosin but also by the CXCR2-specific allosteric inverse agonist SB265610. Furthermore, Labetalol, which is marketed for hypertension as a nonselective β-adrenoceptor antagonist with α(1)AR antagonist properties, was identified as a heteromer-specific-biased agonist exhibiting partial agonism for inositol phosphate production but essentially full agonism for β-arrestin recruitment at the α(1A)AR-CXCR2 heteromer. Finally, bioluminescence resonance energy transfer studies with both receptors tagged suggest that α(1A)AR-CXCR2 heteromerization occurs constitutively and is not modulated by ligand. These findings support the concept of GPCR heteromer complexes exhibiting distinct pharmacology, thereby providing additional mechanisms through which GPCRs can potentially achieve their diverse biological functions. This has important implications for the use and future development of pharmaceuticals targeting these receptors.     

Gene expression of C-type natriuretic peptide and of its specific receptor NPR-B in human leukocytes of healthy and heart failure subjects

C-type natriuretic peptide (CNP), a member of the family of natriuretic peptides, is synthesized and secreted from monocytes and macrophages that resulted to be a source of CNP at inflammatory sites. This suggests that special attention should be focused on the possible role of CNP in the immune system, in addition to its effects on the cardiovascular system. The aim of this study was to evaluate the possibility of measuring the mRNA expression of CNP and NPR-B, its specific receptor, in human whole blood samples of healthy (N; n=7) and heart failure (HF; n=7) subjects by Real-Time PCR (RT-PCR). Total RNA was extracted from leukocytes with QIAamp RNA Blood Kit and/or with PAXgene Blood RNA Kit. RT-PCR was performed and optimized for each primer. The experimental results were normalized with the three most stably expressed genes. CNP and NPR-B expression trend was similar in both fresh and frozen human whole blood. Significant higher levels of CNP and NPR-B mRNA expression were found in HF patients with respect to controls (CNP: N=1.23±0.33 vs. HF=6.54±2.09 p=0.027; NPR-B: N=0.85±0.23 vs. HF=5.31±1.98 p=0.04). A significant correlation between CNP and NPR-B (r=0.86, p<0.0001) was observed. Further studies are needed to clarify the pathophysiological properties of this peptide but the possibility to measure CNP and NPR-B mRNA expression in human leukocytes with a fast and easy procedure is a useful starting point for future investigation devoted to better understand the biomolecular processes associated to different diseases.     

Developments in clinical cell therapy

Immunotherapy has become an important part of hematopoietic stem cell (HSC) transplantation and cancer therapy. Regenerative and reparative properties of somatic cell-based therapies hold tremendous promise for repairing injured tissue, preventing and reversing damage to organs, and restoring balance to compromised immune systems. The principles and practices of the diverse aspects of immune therapy for cancer, HSC transplantation and regenerative medicine have many commonalities. This meeting report summarizes a workshop sponsored by the National Heart, Lung and Blood Institute (NHLBI) and Production Assistance for Cellular Therapies (PACT), held on 23–24 April 2009 at the National Institutes of Health (NIH, USA). A series of scientific sessions and speakers highlighted key aspects of the latest scientific, clinical and technologic developments in cell therapy, involving a unique set of cell products with a special emphasis on converging concepts in these fields.     

Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response

We have previously shown that a single portal vein infusion of a recombinant adeno-associated viral vector (rAAV) expressing canine Factor IX (F.IX) resulted in long-term expression of therapeutic levels of F.IX in dogs with severe hemophilia B. We carried out a phase 1/2 dose-escalation clinical study to extend this approach to humans with severe hemophilia B. rAAV-2 vector expressing human F.IX was infused through the hepatic artery into seven subjects. The data show that: (i) vector infusion at doses up to 2 x 10(12) vg/kg was not associated with acute or long-lasting toxicity; (ii) therapeutic levels of F.IX were achieved at the highest dose tested; (iii) duration of expression at therapeutic levels was limited to a period of approximately 8 weeks; (iv) a gradual decline in F.IX was accompanied by a transient asymptomatic elevation of liver transaminases that resolved without treatment. Further studies suggested that destruction of transduced hepatocytes by cell-mediated immunity targeting antigens of the AAV capsid caused both the decline in F.IX and the transient transaminitis. We conclude that rAAV-2 vectors can transduce human hepatocytes in vivo to result in therapeutically relevant levels of F.IX, but that future studies in humans may require immunomodulation to achieve long-term expression.     

Temporal mismatch between induction of superoxide dismutase and ascorbate peroxidase correlates with high H2O2 concentration in seawater from clofibrate-treated red algae Kappaphycus alvarezii

Algal cells have developed different strategies to cope with the common environmentally promoted generation of H(2)O(2), which include induction of catalase (CAT) and ascorbate peroxidase (APX), massive H(2)O(2) release in seawater, and synthesis of volatile halocarbons by specific peroxidases. The antioxidant adaptability of the economically important carrageenophyte Kappaphycus alvarezii (Doty) Doty (Gigartinales: Rhodophyta) was tested here against exposure to clofibrate (CFB), a known promoter of peroxisomal beta-oxidation in mammals and plants. Possibly as a consequence of CFB-induced H2O2 peroxisomal production, the maximum concentration of H(2)O(2) in the seawater of red algae cultures was found to occur (120+/-17 min) after the addition of CFB, which was followed by a significant decrease in the photosynthetic activity of PSII after 24 h. Interestingly, 4 h after the addition of CFB, the total SOD activity was about 2.5-fold higher than in the control, whereas no significant changes were observed in lipoperoxidation levels (TBARS) or in CAT and APX activities. The two H(2)O(2)-scavenging enzymes were only induced later (after 72 h), whereupon CAT showed a dose-dependent response with increasing concentrations of CFB. A more pronounced increase of TBARS concentration than in the controls was evidenced when a 50 microM Fe(2+/3+) solution (3:2 ratio) was added to CFB-treated cultures, suggesting that the combination of exacerbated H(2)O(2) levels in the seawater-in this work, caused by CFB exposure-and Fenton-reaction catalyst (ferric/ferrous ions), imposes harsh oxidative conditions on algal cultures. The bulk of data suggests that K. alvarezii possesses little ability to promptly induce CAT and APX compared to the immediately responsive antioxidant enzyme SOD and, to avoid harmful accumulation of H(2)O(2), the red alga presumably releases H(2)O(2) into the surrounding medium as an alternative mechanism.     

High-affinity HLA-A(*)02.01 peptides from parathyroid hormone-related protein generate in vitro and in vivo antitumor CTL response without autoimmune side effects

Parathyroid hormone-related protein (PTH-rP), a protein produced by prostate carcinoma and other epithelial cancers, is a key agent in the development of bone metastases. We investigated whether the protein follows the self-tolerance paradigm or can be used as a target Ag for anticancer immunotherapy by investigating the immunogenicity of two HLA-A(*)02.01-binding PTH-rP-derived peptides (PTR-2 and -4) with different affinity qualities. PTH-rP peptide-specific CTL lines were generated from the PBMC of two HLA-A(*)02.01(+) healthy individuals, stimulated in vitro with PTH-rP peptide-loaded autologous dendritic cells and IL-2. The peptide-specific CTLs were able to kill PTH-rP(+)HLA-A(*)02.01(+) breast and prostate carcinoma cell lines. The two peptides were also able to elicit a strong antitumor PTH-rP-specific CTL response in HLA-A(*)02.01 (HHD) transgenic mice. The vaccinated mice did not show any sign of side effects due to cell-mediated autoimmunity or toxicity. In this study we describe two immunogenic and toxic-free PTH-rP peptides as valid candidates for the design of peptide-based vaccination strategies against prostate cancer and bone metastases from the most common epithelial malignancies.     

Effect of the globus pallidus on hippocampal epilepsy in the cat

The action of the pallidum on electrically induced afterdischarge of the hippocampus (HAD) was studied. Significant facilitatory influences on HAD duration were observed when pallidal conditional stimulation immediately preceded hippocampal test stimulation. The phenomenon appeared to be into correlation with the interval between conditioning and test stimulation. Experimental data are discussed as strongly presumptive of a functional interrelationship between the inhibitory role played by the caudate on both pallidum and hippocampus; facilitatory influence of the pallidum on HAD duration is discussed too.