Peptide-Conjugated Nanoparticles Reduce Positive Co-stimulatory Expression and T Cell Activity to Induce Tolerance.

Targeted approaches to treat autoimmune diseases would improve upon current therapies that broadly suppress the immune system and lead to detrimental side effects. Antigen-specific tolerance was induced using poly(lactide-co-glycolide) nanoparticles conjugated with disease-relevant antigen to treat a model of multiple sclerosis. Increasing the nanoparticle dose and amount of conjugated antigen both resulted in more durable immune tolerance. To identify active tolerance mechanisms, we investigated downstream cellular and molecular events following nanoparticle internalization by antigen-presenting cells. The initial cell response to nanoparticles indicated suppression of inflammatory signaling pathways. Direct and functional measurement of surface MHC-restricted antigen showed positive correlation with both increasing particle dose from 1 to 100 µg/mL and increasing peptide conjugation by 2-fold. Co-stimulatory analysis of cells expressing MHC-restricted antigen revealed most significant decreases in positive co-stimulatory molecules (CD86, CD80, and CD40) following high doses of nanoparticles with higher peptide conjugation, whereas expression of a negative co-stimulatory molecule (PD-L1) remained high. T cells isolated from mice immunized against myelin proteolipid protein (PLP139-151) were co-cultured with antigen-presenting cells administered PLP139-151-conjugated nanoparticles, which resulted in reduced T cell proliferation, increased T cell apoptosis, and a stronger anti-inflammatory response. These findings indicate several potential mechanisms used by peptide-conjugated nanoparticles to induce antigen-specific tolerance.

Intravenous delivery of adeno-associated viral vector serotype 9 mediates effective gene expression in ischemic stroke lesion and brain angiogenic foci.

BACKGROUND AND PURPOSE: Adeno-associated viral vector (AAV) is a powerful tool for delivering genes to treat brain diseases. Intravenous delivery of a self-complementary but not single-stranded AAV9 (ssAAV9) mediates robust gene expression in the adult brain. We tested if ssAAV9 effectively mediates gene expression in the ischemic stroke lesion and angiogenic foci. METHODS: Focal ischemic stroke was induced by permanent occlusion of the left middle cerebral artery (MCAO) and focal angiogenesis was induced by injecting an AAV expressing vascular endothelial growth factor (AAV-VEGF) into the basal ganglia. ssAAV vectors that have cytomegalovirus (CMV) promoter driving (AAV-CMVLacZ) or hypoxia response elements controlling (AAV-H9LacZ) LacZ expression were packaged in AAV9 or AAV1 capsid and injected into mice through the jugular vein 1 hour after MCAO or 4 weeks after the induction of angiogenesis. LacZ gene expression was analyzed in the brain and other organs 5 days after LacZ vector injection. RESULTS: LacZ expression was detected in the peri-infarct region of AAV9-CMVLacZ and AAV9-H9LacZ-injected MCAO mice and the brain angiogenic foci of AAV9-CMVLacZ-injected mice. Minimum LacZ expression was detected in the brain of AAV1-CMVLacZ-injected mice. Robust LacZ expression was found in the liver and heart of AAV-CMVLacZ-injected mice, but not in AAV9-H9LacZ-injected mice. CONCLUSIONS: ssAAV9 could be a useful tool to deliver therapeutic genes to the ischemic stroke lesion or brain angiogenic foci.

Endoglin deficiency in bone marrow is sufficient to cause cerebrovascular dysplasia in the adult mouse after vascular endothelial growth factor stimulation.

BACKGROUND AND PURPOSE: Bone marrow-derived cells (BMDCs) home to vascular endothelial growth factor (VEGF)-induced brain angiogenic foci, and VEGF induces cerebrovascular dysplasia in adult endoglin heterozygous (Eng(+/-)) mice. We hypothesized that Eng(+/-) BMDCs cause cerebrovascular dysplasia in the adult mouse after VEGF stimulation. METHODS: BM transplantation was performed using adult wild-type (WT) and Eng(+/-) mice as donors/recipients. An adeno-associated viral vector expressing VEGF was injected into the basal ganglia 4 weeks after transplantation. Vascular density, dysplasia index (vessels >15 µm/100 vessels), and BMDCs in the angiogenic foci were analyzed. RESULTS: The dysplasia index of WT/Eng(+/-) BM mice was higher than WT/WT BM mice (P<0.001) and was similar to Eng(+/-)/Eng(+/-) BM mice (P=0.2). Dysplasia in Eng(+/-) mice was partially rescued by WT BM (P<0.001). WT/WT BM and WT/Eng(+/-) BM mice had similar numbers of BMDCs in the angiogenic foci (P=0.4), most of which were CD68(+). Eng(+/-) monocytes/macrophages expressed less matrix metalloproteinase-9 and Notch1. CONCLUSIONS: Endoglin-deficient BMDCs are sufficient for VEGF to induce vascular dysplasia in the adult mouse brain. Our data support a previously unrecognized role of BM in the development of cerebrovascular malformations.

Design of biodegradable nanoparticles to modulate phenotypes of antigen-presenting cells for antigen-specific treatment of autoimmune disease.

Current therapeutic options for autoimmune diseases, such as multiple sclerosis (MS), often require lifelong treatment with immunosuppressive drugs, yet strategies for antigen-specific immunomodulation are emerging. Biodegradable particles loaded with disease-specific antigen, either alone or with immunomodulators, have been reported to ameliorate disease. Herein, we hypothesized that the carrier could impact polarization of the immune cells that associate with particles and the subsequent disease progression. Single injection of three polymeric carriers, 50:50 poly (DL-lactide-co-glycolide) (PLG) with two molecular weights (Low, High) and poly (DL-lactide) (PLA), loaded with the disease-specific antigen, proteolipid protein (PLP139-151), were investigated for the ability to attenuate clinical scores in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. At a low particle dose, mice treated with PLA-based particles had significantly lower clinical scores at the chronic stage of the disease over 200 days post immunization, while neither PLG-based particles nor OVA control particles reduced the clinical scores. Compared to PLG-based particles, PLA-based particles were largely associated with Kupffer cells and liver sinusoidal endothelial cells, which had a reduced co-stimulatory molecule expression that correlated with a reduction of CD4+ T-cell populations in the central nervous system. Delivery of PLA-based particles encapsulated with higher levels of PLP139-151 at a reduced dose were able to completely ameliorate EAE over 200 days along with inhibition of Th1 and Th17 polarization. Collectively, our study demonstrates that the carrier properties and antigen loading determine phenotypes of immune cells in the peripheral organs, influencing the amelioration of both acute and chronic stages of autoimmunity.

Designing drug-free biodegradable nanoparticles to modulate inflammatory monocytes and neutrophils for ameliorating inflammation.

Inflammation associated with autoimmune diseases and chronic injury is an initiating event that leads to tissue degeneration and dysfunction. Inflammatory monocytes and neutrophils systemically circulate and enter inflamed tissue, and pharmaceutical based targeting of these cells has not substantially improved outcomes and has had side effects. Herein, we investigated the design of drug-free biodegradable nanoparticles, notably without any active pharmaceutical ingredient or targeting ligand, that target circulating inflammatory monocytes and neutrophils in the vasculature to inhibit them from migrating into inflamed tissue. Nanoparticles were formed from 50:50 poly(DL-lactide-co-glycolide) (PLG) with two molecular weights (Low, High) and poly(DL-lactide) (PLA) (termed PLG-L, PLG-H, and PDLA, respectively) and were analyzed for their association with monocytes and neutrophils and their impact on disease course along with immune cell trafficking. For particles injected intravenously for 6 consecutive days to mice with experimental autoimmune encephalomyelitis (EAE), PLG-H particles had significantly lower EAE clinical scores than PBS control, while PLG-L and PDLA particles had modest or negligible effect on EAE onset. In vivo and in vitro data suggests that PLG-H particles had high association with immune cells, with preferential association with blood neutrophils relative to other particles. PLG-H particles restrained immune cells from the central nervous system (CNS), with increased accumulation in the spleen, which was not observed for mice receiving PDLA or control treatments. These results demonstrate that the particle composition influences the association with inflammatory monocytes and neutrophils in the vasculature, with the potential to redirect trafficking and ameliorate inflammation.

Apoptosis-induced CXCL5 accelerates inflammation and growth of prostate tumor metastases in bone.

During tumor progression, immune system phagocytes continually clear apoptotic cancer cells in a process known as efferocytosis. However, the impact of efferocytosis in metastatic tumor growth is unknown. In this study, we observed that macrophage-driven efferocytosis of prostate cancer cells in vitro induced the expression of proinflammatory cytokines such as CXCL5 by activating Stat3 and NF-κB(p65) signaling. Administration of a dimerizer ligand (AP20187) triggered apoptosis in 2 in vivo syngeneic models of bone tumor growth in which apoptosis-inducible prostate cancer cells were either coimplanted with vertebral bodies, or inoculated in the tibiae of immunocompetent mice. Induction of 2 pulses of apoptosis correlated with increased infiltration of inflammatory cells and accelerated tumor growth in the bone. Apoptosis-induced tumors displayed elevated expression of the proinflammatory cytokine CXCL5. Likewise, CXCL5-deficient mice had reduced tumor progression. Peripheral blood monocytes isolated from patients with bone metastasis of prostate cancer were more efferocytic compared with normal controls, and CXCL5 serum levels were higher in metastatic prostate cancer patients relative to patients with localized prostate cancer or controls. Altogether, these findings suggest that the myeloid phagocytic clearance of apoptotic cancer cells accelerates CXCL5-mediated inflammation and tumor growth in bone, pointing to CXCL5 as a potential target for cancer therapeutics.

Integrin ß8 Deletion Enhances Vascular Dysplasia and Hemorrhage in the Brain of Adult Alk1 Heterozygous Mice.

Brain arteriovenous malformation (bAVM), characterized by tangled dysplastic vessels, is an important cause of intracranial hemorrhage in young adults, and its pathogenesis and progression are not fully understood. Patients with haploinsufficiency of transforming growth factor-ß (TGF-ß) receptors, activin receptor-like kinase 1 (ALK1) or endoglin (ENG) have a higher incidence of bAVM than the general population. However, bAVM does not develop effectively in mice with the same haploinsufficiency. The expression of integrin ß8 subunit (ITGB8), another member in the TGF-ß superfamily, is reduced in sporadic human bAVM. Brain angiogenic stimulation results at the capillary level of vascular malformation in adult Alk1 haploinsufficient (Alk1 +/- ) mice. We hypothesized that deletion of Itgb8 enhances bAVM development in adult Alk1 +/- mice. An adenoviral vector expressing Cre recombinase (Ad-Cre) was co-injected with an adeno-associated viral vector expressing vascular endothelial growth factor (AAV-VEGF) into the brain of Alk1 +/-;Itgb8-floxed mice to induce focal Itgb8 gene deletion and angiogenesis. We showed that compared with Alk +/- mice (4.75 ± 1.38/mm2), the Alk1 +/-;Itgb8-deficient mice had more dysplastic vessels in the angiogenic foci (7.14 ± 0.68/mm2, P = 0.003). More severe hemorrhage was associated with dysplastic vessels in the brain of Itgb8-deleted Alk1 +/- , as evidenced by larger Prussian blue-positive areas (1278 ± 373 pixels/mm2 vs. Alk1 +/-  : 320 ± 104 pixels/mm2; P = 0.028). These data indicate that both Itgb8 and Alk1 are important in maintaining normal cerebral angiogenesis in response to VEGF. Itgb8 deficiency enhances the formation of dysplastic vessels and hemorrhage in Alk1 +/- mice.

Nongenetic factors associated with stress urinary incontinence.

OBJECTIVE: To explore the role of hereditary and environmental factors on the development of stress urinary incontinence in a large cohort of identical and nonidentical twins. METHODS: This is a large, population-based, classic twin study of twin sisters recruited to complete a health survey at the world's largest annual twins festival during 2003-2008. Concordance rates were calculated and structural equation models were used to estimate the contribution of genetic effects compared with environmental factors toward the development of stress urinary incontinence. RESULTS: Eight hundred eighty-two twin sister pairs (n=1,764), including 765 identical and 117 nonidentical twin sister pairs, completed the questionnaires. Sequential structural equation modeling revealed that common environmental factors contributed 77.6% (95% confidence interval [CI], 41.4-83.8; P<.001) of the variance and unique environmental factors contributed 20.9% (95% CI, 15.8-26.7; P<.001) of the variance. The effect of genetics was not statistically significant at 1.49% (95% CI, 0.0-38.8; P=.46). CONCLUSION: Female stress urinary incontinence is more a consequence of environmental risk factors than heredity. This epidemiologic insight should be considered in preventive health efforts. LEVEL OF EVIDENCE: II.