An alternative splicing variant in Clcn7-/- mice prevents osteopetrosis but not neural and retinal degeneration.

The ubiquitously expressed chloride channel 7 (CLCN7) is present within the ruffled border of osteoclasts. Mutations in the CLCN7 gene in humans (homologous to murine Clcn7) are responsible for several types of osteopetrosis in humans, and deficiencies in CLCN7 can present with retinal degeneration and a neuronal storage disease. A previously reported Clcn7(-/-) mouse showed diffuse osteopetrosis accompanied by severe retinal and neuronal degeneration. In contrast, the authors produced a novel Clcn7(-/-) mutant where mice did not develop osteopetrosis but still developed lethal neural and retinal degeneration. In these mice, there was a rapid progressive loss of the outer nuclear layer and photoreceptor layers of the retina. Laminar degeneration and necrosis of neurons in layers IV and V of the cerebral cortex and in the CA2/CA3 regions of the hippocampus were associated with intraneuronal accumulations of autofluorescent granules (periodic acid-Schiff positive). The extensive reactive gliosis was always associated with the accumulation of intraneuronal cytoplasmic material. The authors found, through quantitative real time polymerase chain reaction analyses, that an alternate Clcn7 transcript (previously identified only in bone marrow) showed minimal expression in the brain and eye but moderate expression in bone, which correlates with rescue of the osteopetrotic phenotype in the face of continued retinal and neuronal degeneration. Findings in this knockout mouse model prove that osteopetrotic compression of the brain is not responsible for neuronal and retinal degeneration in CLCN7-deficient mice; rather, they suggest that neurotoxicity is most likely due to lysosomal dysfunction as a result of the functional lack of this chloride channel in the central nervous system and eye.

PD-L1 expression is regulated by both DNA methylation and NF-kB during EMT signaling in non-small cell lung carcinoma.

Tumor cells, which undergo Epithelial-mesenchymal transition (EMT) acquire increased capacities of proliferation, invasion and have the ability to generate metastases by escaping the immune system during their systemic migration. To escape the immune system, cancer cells may induce tolerance or resist elimination by immune effectors via multiple mechanisms and we hypothesized that EMT may control the expression of immune checkpoint inhibitors, then promoting immune evasion. PD-L1 (programmed cell death ligand 1) but not PD-L2 nor Galectin 9 or Death receptor (DR4, DR5 and Fas) and ligands (FasL and TRAIL) expression was up-regulated during cytokine-driven EMT in a reversible manner. Moreover PD-L1 is overexpressed in VIMENTIN positive NSCLC tissues. We also demonstrated that the expression of PD-L1 required both TNFα and TGFß1. Indeed, TGFß1 decreased DNMT1 content and that resulted in PD-L1 promoter demethylation whereas TNFα induced the NF-κB pathway that promoted expression of demethylated PD-L1 promoter.

LFA-1 is sufficient in mediating neutrophil emigration in Mac-1-deficient mice.

To better define the specific function of Mac-1 (CD11b) versus LFA-1 (CD11a) and the other CD11 integrins in vivo, we have disrupted murine CD11b by targeted homologous recombination in embryonic stem cells and generated mice which are homozygous for a mutation in CD11b. A null mutation was confirmed by Southern blotting, RNase protection assay, immunohistochemistry, and flow cytometry. Neutrophils isolated from mice deficient in Mac-1 were defective in adherence to keyhole limpet hemocyanin-coated glass, iC3b-mediated phagocytosis, and homotypic aggregation. When challenged by thioglycollate intraperitoneally, Mac-1-deficient mice had similar levels of neutrophil accumulation in the peritoneal cavity at 1, 2, and 4 h. Treatment with mAb to LFA-1 blocked 78% of neutrophil accumulation in Mac-1-deficient mice and 58% in wild-type mice. Neutrophil emigration into the peritoneal cavity 16 h after the implantation of fibrinogen-coated disks was not reduced in Mac-1-deficient mice whereas neutrophil adhesion to the fibrinogen-coated disks was reduced by > 90%. Neutrophils from Mac-1-deficient mice also showed reduced degranulation. Our results demonstrate that Mac-1 plays a critical role in mediating binding of neutrophils to fibrinogen and neutrophil degranulation, but is not necessary for effective neutrophil emigration, which is more dependent upon LFA-1.

Safety toxicity study of plasmid-based IL-12 therapy in Cynomolgus monkeys.

We have investigated the potential toxicity of hlL-12 DNA plasmid formulated with 5% polyvinylpyrrolidone (PVP) administered twice weekly via subcutaneous injections to Cynomolgus monkeys for four weeks, and have evaluated recovery from any effects of the test article over a four-week treatment-free period. Doses of the formulated hIL-12 plasmid were selected based on anti-tumour efficacy studies previously conducted in mice. The duration of the study and the frequency of dosing were designed to support clinical trials. No clinical signs indicative of an adverse effect of administration of formulated hIL-12 plasmid were observed. There were no apparent effects of the formulated hIL-12 plasmid on body weights or on serum chemistry, haematology, coagulation or urinalysis parameters. No treatment-elated ocular abnormalities were evident. In addition, examination of the electrocardiograms from all monkeys at the pre-study, week-4, and week-8 time points did not reveal any treatment-related effects. No treatment-related gross lesions were noted at days 28 or 57. Slight histopathological changes associated with high doses of PVP vehicle were observed at both time points. These results suggested that the administration of formulated hIL-12 plasmid at a dose level up to 18 mg kg(-1) dose twice per week for four weeks to experimentally naïve Cynomolgus monkeys did not result in significant toxicity. These results support further testing of this gene therapy in clinical trials.

Poly-L-glutamate, an anionic polymer, enhances transgene expression for plasmids delivered by intramuscular injection with in vivo electroporation.

Intramuscular (i.m.) injection of plasmids followed by electropermeabilization is an efficient process to deliver genes into skeletal myofibers that permits proteins to be produced and secreted at therapeutically relevant levels. To further improve skeletal muscle as a bioreactor, we identified a formulation that elevates transgene expression in myofibers after i.m. injection and electroporation. With secreted placental alkaline phosphate (SEAP) as reporter gene, plasmid formulated with poly-L-glutamate produced two- to eight-fold higher levels of SEAP in mouse serum than plasmid in saline. Various concentrations and molecular weights of poly-L-glutamate were similarly effective, but 6 mg/ml of 15-50 kDa poly-L-glutamate consistently yielded the highest expression levels. The poly-L-glutamate formulation was effective in two different muscle groups in mice at various plasmid doses for several transgenes, including an erythropoietin (EPO) gene, for which expression was elevated four- to 12-fold in comparison to animals that received EPO plasmid in saline. Transgene expression was localized to myofibers. Poly-L-glutamate may improve transgene expression in part by increasing plasmid retention in skeletal muscle. Poly-L-glutamate did not enhance gene transfer in the absence of electroporation. Therefore, the polymer is a novel formulation that specifically enhances the transfer and expression of genes delivered with electroporation.

Ligand-dependent regulation of vascular endothelial growth factor and erythropoietin expression by a plasmid-based autoinducible GeneSwitch system.

We investigated the ability of an improved mifepristone-dependent GeneSwitch system to regulate the expression of genes for two therapeutic proteins: vascular endothelial growth factor (VEGF) and erythropoietin. The GeneSwitch system consisted of two plasmids, one encoding the chimeric GeneSwitch protein, the other an inducible transgene. When the constitutive CMV promoter of the GeneSwitch plasmid was replaced by an autoinducible promoter consisting of four copies of GAL4 DNA binding sites linked to a minimal thymidine kinase promoter, the tightness of transgene regulation was improved by an order of magnitude. Quantitative RT-PCR analysis of GeneSwitch mRNA confirmed that the autoinducible promoter was responsive to mifepristone. We demonstrated the ability of the improved GeneSwitch system to regulate the expression of VEGF or erythropoietin in a biologically relevant manner after delivery of plasmids to the hind-limb muscle of adult mice. This ability of the autoinducible GeneSwitch system to regulate the expression of therapeutic proteins in mice indicates its potential for use in human gene therapy applications.

Molecular evidence for induction of intracellular adhesion molecule-1 in the viable border zone associated with ischemia-reperfusion injury of the dog heart.

BACKGROUND: Acute inflammation may play a role in injury during reperfusion following myocardial ischemia. Studies in vitro suggest that intracellular adhesion molecule-1 (ICAM-1) mediates neutrophil adherence to cardiac myocytes and neutrophil-mediated injury. We have shown cytokine activity in postischemic cardiac lymph sufficient to maximally express ICAM-1 on myocytes and that ICAM-1 mRNA is found in the previously ischemic myocardium early in reperfusion. METHODS AND RESULTS: In the present study, we used in situ hybridization techniques to detect ICAM-1 mRNA and examine the cells of origin, relation to cell injury, and relation to inflammatory infiltration after 1 hour of ischemia and varying times of reperfusion. By 1 hour of reperfusion, ICAM-1 mRNA was detected in much of the ischemic myocardium, except in areas of contraction band necrosis. At 2 and 3 hours, a clear demarcation of necrotic areas surrounding ischemic areas of viable myocardium with ICAM-1 mRNA staining was present, and ICAM-1 mRNA staining increased with time. Nonischemic areas had no visible ICAM-1 mRNA staining in the first 3 hours. By 24 hours of reperfusion, ICAM-1 mRNA was present in both control and ischemic segments (excluding the necrotic areas) compatible with a generalized circulation of cytokines persistent at 24 hours. In the absence of reperfusion, ICAM-1 mRNA staining was not seen in the first 3 hours and was markedly reduced at 24 hours. The interface of viable and necrotic cells also contained the most extensive inflammatory infiltration. CONCLUSIONS: Evidence is presented that induction of ICAM-1 mRNA has highly specific localization to ischemic but viable myocardium. Induction of ICAM-1 mRNA transcription in early reperfusion may render the viable "border zone" susceptible to neutrophil-induced injury.

Molecular evidence for a border zone vulnerable to inflammatory reperfusion injury.

Acute inflammation has been suggested as a potential mechanism for some of the injury associated with reperfusion of the ischemic myocardium. This hypothesis implies that viable myocardial cells adjacent to the lethally injured cells are vulnerable to injury induced by the neutrophil influx observed to attend reperfusion. In our previous work, we demonstrated that the presence of ICAM-1 on the surface of cardiac myocytes is required for neutrophils to directly damage them; blocking monoclonal antibodies to either ICAM-1 on cardiac myocytes or Mac-1 on activated neutrophils completely precluded neutrophil-induced myocyte injury. We also demonstrated that postischemic cardiac lymph (cardiac extracellular fluid) contained leukotactic factors (primarily C5a) and cytokines present in concentrations sufficient to maximally induce Mac-1 on the surface of neutrophils and ICAM-1 on the surface of isolated dog cardiac myocytes. The present study sought to further these observations by examining the site of potential ICAM-1 induction as a function of time of reperfusion, degree of ischemia, and viability of myocardial cells. Our evidence suggests that ICAM-1 mRNA is induced very early after reperfusion only in the previously ischemic myocardium and is not seen in the nonischemic myocardium during the early hours of reperfusion. Moreover, ICAM-1 mRNA induction is seen most intensely in the ischemic area directly bordering the necrotic area (which, after 1-hr reperfusion, does not contain any ICAM-1 mRNA) and immediately abutting the site of maximal influx of neutrophils. Thus, the induction of ICAM-1 and the influx of neutrophils (presumably activated by the chemotactic factors that guided their migration) exists on the border between viable and necrotic cells. This provides the first direct molecular evidence for a jeopardized border zone on the edge of myocardial infarction during reperfusion. As previously demonstrated, this reaction is wholly dependent upon tissue injury of the ischemic myocardium and therefore represents an example of a mechanism of injury extension induced as a reaction to a primary injury. The degree of specificity of this reaction demonstrated by the subendocardial sparing directly adjacent to ischemic cells suggests finely modulated mechanisms by which this process is controlled.

Stem cell factor induction is associated with mast cell accumulation after canine myocardial ischemia and reperfusion.

BACKGROUND: Myocardial infarction is associated with an intense inflammatory reaction leading to healing and scar formation. Because mast cells are a significant source of fibrogenic factors, we investigated mast cell accumulation and regulation of stem cell factor (SCF), a potent growth and tactic factor for mast cells, in the healing myocardium. METHODS AND RESULTS: Using a canine model of myocardial ischemia and reperfusion, we demonstrated a striking increase of mast cell numbers during the healing phase of a myocardial infarction. Mast cell numbers started increasing after 72 hours of reperfusion, showing maximum accumulation in areas of collagen deposition (12.0+/-2.6-fold increase; P<0.01) and proliferating cell nuclear antigen (PCNA) expression. The majority of proliferating cells were identified as alpha-smooth muscle actin-positive myofibroblasts or factor VIII-positive endothelial cells. Mast cells did not appear to proliferate. Using a nuclease protection assay, we demonstrated induction of SCF mRNA within 72 hours of reperfusion. Immunohistochemical studies demonstrated that a subset of macrophages was the source of SCF immunoreactivity in the infarcted myocardium. SCF protein was not found in endothelial cells and myofibroblasts. Intravascular tryptase-positive, FITC-avidin-positive, CD11b-negative mast cell precursors were noted in the area of healing and in the cardiac lymph after 48 to 72 hours of reperfusion. CONCLUSIONS: Mast cells increase in number in areas of collagen deposition and PCNA expression after myocardial ischemia. The data provide evidence of mast cell precursor infiltration into the areas of cellular injury. SCF is induced in a subset of macrophages infiltrating the healing myocardium. We suggest an important role for SCF in promoting chemotaxis and growth of mast cell precursors in the healing heart.

Relative contribution of LFA-1 and Mac-1 to neutrophil adhesion and migration.

To differentiate the unique and overlapping functions of LFA-1 and Mac-1, LFA-1-deficient mice were developed by targeted homologous recombination in embryonic stem cells, and neutrophil function was compared in vitro and in vivo with Mac-1-deficient, CD18-deficient, and wild-type mice. LFA-1-deficient mice exhibit leukocytosis but do not develop spontaneous infections, in contrast to CD18-deficient mice. After zymosan-activated serum stimulation, LFA-1-deficient neutrophils demonstrated activation, evidenced by up-regulation of surface Mac-1, but did not show increased adhesion to purified ICAM-1 or endothelial cells, similar to CD18-deficient neutrophils. Adhesion of Mac-1-deficient neutrophils significantly increased with stimulation, although adhesion was lower than for wild-type neutrophils. Evaluation of the strength of adhesion through LFA-1, Mac-1, and CD18 indicated a marked reduction in firm attachment, with increasing shear stress in LFA-1-deficient neutrophils, similar to CD18-deficient neutrophils, and only a modest reduction in Mac-1-deficient neutrophils. Leukocyte influx in a subcutaneous air pouch in response to TNF-alpha was reduced by 67% and 59% in LFA-1- and CD18-deficient mice but increased by 198% in Mac-1-deficient mice. Genetic deficiencies demonstrate that both LFA-1 and Mac-1 contribute to adhesion of neutrophils to endothelial cells and ICAM-1, but adhesion through LFA-1 overshadows the contribution from Mac-1. Neutrophil extravasation in response to TNF-alpha in LFA-1-deficient mice dramatically decreased, whereas neutrophil extravasation in Mac-1-deficient mice markedly increased.