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Paul D Robbins

Publications and source records attributed to Paul D Robbins.

At least 19 recordsLinked to original sources
PubMed2026

Senotypes define the diverse landscape of senescent cells.

Cellular senescence was initially defined in vitro as a stable cell-cycle arrest that occurs after repeated replication, but it is now recognized as a heterogeneous state shaped by cell type, species, senescence-inducing stress, tissue microenvironment and time. To organize this complexity, we propose the term 'senotype' to classify senescent cells by their inputs, molecular features and functional effects. We outline a practical framework incorporating: (1) cell identity and context; (2) inducing mechanism; (3) temporal stage; (4) multimodal molecular and structural features; and (5) physiological or pathological functions. Experimentally defined senotypes can serve as references for interpreting tissue-derived senotypes, where parameters may be incomplete. Senotypes should be anchored in combinations of core hallmarks (that is, durable cell-cycle arrest, altered secretory profiles, macromolecular or organelle damage, disrupted homeostasis) rather than single markers. Advances in single-cell, spatial, proteomic and computational methods enable rigorous senotype characterization, improving consistency and accelerating development of targeted senotherapeutics.

Cellular Senescence↗
PubMed2024

The Ercc1-/Δ mouse model of XFE progeroid syndrome undergoes accelerated retinal degeneration.

Age-related macular degeneration (AMD) is a major cause of vision loss in older adults. AMD is caused by degeneration in the macula of the retina. The retina is the highest oxygen consuming tissue in our body and is prone to oxidative damage. DNA damage is one hallmark of aging implicated in loss of organ function. Genome instability has been associated with several disorders that result in premature vision loss. We hypothesized that endogenous DNA damage plays a causal role in age-related retinal changes. To address this, we used a genetic model of systemic depletion of expression of the DNA repair enzyme ERCC1-XPF. The neural retina and retinal pigment epithelium (RPE) from Ercc1-/Δ mice, which models a human progeroid syndrome, were compared to age-matched wild-type (WT) and old WT mice. By 3-months-of age, Ercc1-/Δ mice presented abnormal optokinetic and electroretinogram responses consistent with photoreceptor dysfunction and visual impairment. Ercc1-/Δ mice shared many ocular characteristics with old WT mice including morphological changes, elevated DNA damage markers (γ-H2AX and 53BP1), and increased cellular senescence in the neural retinal and RPE, as well as pathological angiogenesis. The RPE is essential for the metabolic health of photoreceptors. The RPE from Ercc1-/Δ mice displayed mitochondrial dysfunction causing a compensatory glycolytic shift, a characteristic feature of aging RPE. Hence, our study suggests spontaneous endogenous DNA damage promotes the hallmarks of age-related retinal degeneration.

Animals↗
PubMed2020

Tissue specificity of senescent cell accumulation during physiologic and accelerated aging of mice.

Senescent cells accumulate with age in vertebrates and promote aging largely through their senescence-associated secretory phenotype (SASP). Many types of stress induce senescence, including genotoxic stress. ERCC1-XPF is a DNA repair endonuclease required for multiple DNA repair mechanisms that protect the nuclear genome. Humans or mice with reduced expression of this enzyme age rapidly due to increased levels of spontaneous, genotoxic stress. Here, we asked whether this corresponds to an increased level of senescent cells. p16Ink4a and p21Cip1 mRNA were increased ~15-fold in peripheral lymphocytes from 4- to 5-month-old Ercc1-/∆ and 2.5-year-old wild-type (WT) mice, suggesting that these animals exhibit a similar biological age. p16Ink4a and p21Cip1 mRNA were elevated in 10 of 13 tissues analyzed from 4- to 5-month-old Ercc1-/∆ mice, indicating where endogenous DNA damage drives senescence in vivo. Aged WT mice had similar increases of p16Ink4a and p21Cip1 mRNA in the same 10 tissues as the mutant mice. Senescence-associated β-galactosidase activity and p21Cip1 protein also were increased in tissues of the progeroid and aged mice, while Lamin B1 mRNA and protein levels were diminished. In Ercc1-/Δ mice with a p16Ink4a luciferase reporter, bioluminescence rose steadily with age, particularly in lung, thymus, and pancreas. These data illustrate where senescence occurs with natural and accelerated aging in mice and the relative extent of senescence among tissues. Interestingly, senescence was greater in male mice until the end of life. The similarities between Ercc1-/∆ and aged WT mice support the conclusion that the DNA repair-deficient mice accurately model the age-related accumulation of senescent cells, albeit six-times faster.

Aging↗
PubMed2006

Comparative analysis of antitumor activity of CD40L, RANKL, and 4-1BBL in vivo following intratumoral administration of viral vectors or transduced dendritic cells.

The tumor necrosis factor (TNF) family comprises a group of ligands that regulate cell proliferation, differentiation, activation, maturation and apoptosis through interaction with the corresponding TNF receptor family members. In this study, we have evaluated whether adenovirus-mediated intratumoral gene transfer of CD40L, RANKL, or 4-1BBL elicits an immune response to established murine MC38 and TS/A tumors. Intratumoral administration of the recombinant adenoviral vectors expressing CD40L, RANKL or 4-1BBL 7 days post-tumor cell inoculation resulted in significant inhibition of MC38 tumor growth for all three ligands when compared with control groups treated with either saline or control adenovirus. However, intratumoral injection of Ad-4-1BBL or Ad-CD40L resulted in a significantly stronger inhibition of TS/A tumor progression than did Ad-RANKL treatment. We also demonstrated that intratumoral administration of dendritic cells (DC) transduced with adenoviral vectors encoding the TNF-related ligands resulted in a significant inhibition of MC38 tumor growth as compared with control groups treated with Ad-LacZ-transduced DC or saline-treated DC. In addition, DC overexpressing CD40L secreted considerably more IL-12 and expressed higher levels of the co-stimulatory molecules, CD80, CD86 and CD40, than did DC overexpressing LacZ, 4-1BBL or RANKL. We have also demonstrated that DC/CD40L, DC/4-1BBL, and DC/RANKL survived significantly longer than control DC or DC infected with the LacZ vector. Taken together, these results demonstrate that adenoviral gene transfer of CD40L, RANKL or 4-1BBL elicit a significant antitumor effect in two different tumor models, with CD40L gene transfer inducing the strongest antitumor effect.

4-1BB Ligand↗
PubMed2006

PhiC31 integrase mediates integration in cultured synovial cells and enhances gene expression in rabbit joints.

BACKGROUND: Gene transfer to synovium in joints has been shown to be an effective approach for treating pathologies associated with rheumatoid arthritis (RA) and related joint disorders. However, the efficiency and duration of gene delivery has been limiting for successful gene therapy for arthritis. The transient gene expression that often accompanies non-viral gene delivery can be prolonged by integration of vector DNA into the host genome. We report a novel approach for non-viral gene therapy to joints that utilizes phage phiC31 integrase to bring about unidirectional genomic integration. METHODS: Rabbit and human synovial cells were co-transfected with a plasmid expressing phiC31 integrase and a plasmid containing the transgene and an attB site. Cells were cultured with or without G418 selection and the number of neo-resistant colonies or eGFP cells determined, respectively. Plasmid rescue, PCR query, and DNA sequence analysis were performed to reveal integration sites in the rabbit and human genomes. For in vivo studies, attB-reporter gene plasmids and a plasmid expressing phiC31 integrase were intra-articularly injected into rabbit knees. Joint sections were used for histological analysis of beta-gal expression, and synovial cells were isolated to measure luciferase expression. RESULTS: We demonstrated that co-transfection of a plasmid expressing phiC31 integrase with a plasmid containing the transgene and attB increased the frequency of transgene expression in rabbit synovial fibroblasts and primary human RA synoviocytes. Plasmid rescue and DNA sequence analysis of plasmid-chromosome junctions revealed integration at endogenous pseudo attP sequences in the rabbit genome, and PCR query detected integration at previously characterized integration sites in the human genome. Significantly higher levels of transgene expression were detected in vivo in rabbit knees after intra-articular injection of attB-reporter gene plasmids and a plasmid expressing phiC31 integrase. CONCLUSION: The ability of phiC31 integrase to facilitate genomic integration in synovial cells and increase transgene expression in the rabbit synovium suggests that, in combination with more efficient DNA delivery methods, this integrase system could be beneficial for treatment of rheumatoid arthritis and other joint disorders.

Animals↗
PubMed2006

Hyperactive Himar1 transposase mediates transposition in cell culture and enhances gene expression in vivo.

The use of nonviral delivery systems results in transient gene expression, in part because of the low efficiency of DNA integration. Previously, vectors based on transposon systems such as Sleeping Beauty have been shown to be able to increase stable transfection efficiencies in cell culture and in animal models. Himar1, a reconstructed active transposon belonging to the Tc1/mariner superfamily, also has been used as a vector for stable gene delivery, but the rate of transposition after transfection is low. In this paper, we evaluate the potential of the hyperactive Himar1 transposase C9, in combination with the Himar1 inverted repeat transposon, as a gene delivery vector. The C9 transposase is a hyperactive mutant of Himar1 with two amino acid substitutions, Q131R and E137K, that result in an increase in activity relative to wild type. Here we demonstrate that cotransfection of the C9 transposase with a Himar1-based vector increases the frequency of stable gene expression in human cells in a transposase concentration-dependent manner. In addition, we establish that C9 transposase mediates integration of the transgene in mammalian cells at a frequency similar to that of Sleeping Beauty under some of the conditions tested. Last, we show significantly higher levels of reporter gene expression in vivo in mouse liver and in synovium of rabbit knee joints after injection of the transposon plasmid expressing the transgene and the C9 transposase. These data suggest that vectors based on the Himar1 transposable element, in conjunction with the hyperactive mutant transposase C9, may be suitable vectors for gene therapy applications.

Animals↗
PubMed2006

Broad cellular immunity with robust memory responses to simian immunodeficiency virus following serial vaccination with adenovirus 5- and 35-based vectors.

Adenovirus serotype 35 (Ad35) is a promising vaccine platform for human immunodeficiency virus (HIV) infection and emerging infectious diseases as it is uncommon in humans worldwide and is distinct from Ad5, the major vaccine serotype for which many individuals have pre-existing immunity. The immunogenicity of a first-generation, replication-competent Ad35-based vaccine was tested in the simian immunodeficiency virus (SIV) rhesus macaque model by evaluating its capacity to boost immunity generated by Ad5-based vectors. A series of four immunizations with replication-defective Ad5 vectors expressing SIVmac239 gag induced high-frequency responses mediated by both CD8(+) and CD4(+) T cells directed against several epitopes. Ad5-specific neutralizing antibody responses that did not neutralize Ad35 were rapidly induced but waned over time. Subsequent immunization with Ad5-based vectors was minimally effective, whereas immunization with Ad35-based vectors generated a strong increase in the frequency of Gag-specific T cells with specificities that were unchanged. While this boosting response was relatively transient, challenge with the distinct pathogenic isolate SIV/DeltaB670 generated robust and selective recall responses to Gag with similar specificities as induced by vaccination that were elevated for 25 weeks relative to controls. Vaccination had measurable albeit minor effects on virus load. Unexpectedly, regional hypervariability within the Gag sequence of SIV/DeltaB670 was associated with mutation of the conserved CD8(+) T-cell epitope CM9 without concurrent flanking mutations and in the absence of immune pressure. These findings support the further development of Ad35 as a vaccine vector, and promote vaccine regimens that utilize serial administration of heterologous adenoviruses.

Adenoviruses, Human↗
PubMed2006

Protection of mice and poultry from lethal H5N1 avian influenza virus through adenovirus-based immunization.

The recent emergence of highly pathogenic avian influenza virus (HPAI) strains in poultry and their subsequent transmission to humans in Southeast Asia have raised concerns about the potential pandemic spread of lethal disease. In this paper we describe the development and testing of an adenovirus-based influenza A virus vaccine directed against the hemagglutinin (HA) protein of the A/Vietnam/1203/2004 (H5N1) (VN/1203/04) strain isolated during the lethal human outbreak in Vietnam from 2003 to 2005. We expressed different portions of HA from a recombinant replication-incompetent adenoviral vector, achieving vaccine production within 36 days of acquiring the virus sequence. BALB/c mice were immunized with a prime-boost vaccine and exposed to a lethal intranasal dose of VN/1203/04 H5N1 virus 70 days later. Vaccination induced both HA-specific antibodies and cellular immunity likely to provide heterotypic immunity. Mice vaccinated with full-length HA were fully protected from challenge with VN/1203/04. We next evaluated the efficacy of adenovirus-based vaccination in domestic chickens, given the critical role of fowl species in the spread of HPAI worldwide. A single subcutaneous immunization completely protected chickens from an intranasal challenge 21 days later with VN/1203/04, which proved lethal to all control-vaccinated chickens within 2 days. These data indicate that the rapid production and subsequent administration of recombinant adenovirus-based vaccines to both birds and high-risk individuals in the face of an outbreak may serve to control the pandemic spread of lethal avian influenza.

Adenoviridae↗
PubMed2006

Intra-articular injection of recombinant TRAIL induces synovial apoptosis and reduces inflammation in a rabbit knee model of arthritis.

We demonstrated previously that local, intra-articular injection of an adenoviral vector expressing human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in a rabbit knee model of inflammatory arthritis stimulated synovial apoptosis and reduced inflammation. To examine whether intra-articular injection of recombinant chimeric human TRAIL protein (rTRAIL) also induces apoptosis of proliferating rabbit synovium and reduces inflammation, we used an experimental rabbit arthritis model of rheumatoid arthritis, induced by intra-articular introduction of allogeneic fibroblasts genetically engineered to secrete human IL-1beta. Analysis of synovium isolated from the rabbits treated with intra-articular injection of rTRAIL, relative to saline control, showed areas of extensive acellular debris and large fibrous regions devoid of intact cells, similar to adenoviral mediated TRAIL gene transfer. Extensive apoptosis of the synovial lining was demonstrated using TUNEL analysis of the sections, corresponding to the microscopic findings in hematoxylin and eosin staining. In addition, leukocyte infiltration into the synovial fluid of the inflamed knee joints following rTRAIL treatment was reduced more than 50% compared with the saline control. Analysis of the glycosaminoglycan synthetic rate by cultured cartilage using radiolabeled sulfur and cartilage histology demonstrated that rTRAIL did not adversely affect cartilage metabolism and structure. Analysis of serum alanine aminotransferase showed that intra-articular injection of rTRAIL did not have adverse effects on hepatic function. These results demonstrate that intra-articular injection of rTRAIL could be therapeutic for treating pathologies associated with rheumatoid arthritis.

Animals↗
PubMed2006

Human, viral or mutant human IL-10 expressed after local adenovirus-mediated gene transfer are equally effective in ameliorating disease pathology in a rabbit knee model of antigen-induced arthritis.

IL-10 is a Th2 cytokine important for inhibiting cell-mediated immunity while promoting humoral responses. Human IL-10 (hIL-10) has anti-inflammatory, immunosuppressive as well as immunostimulatory characteristics, whereas viral IL-10 (vIL-10), a homologue of hIL-10 encoded by Epstein Barr virus (EBV), lacks several immunostimulatory functions. The immunostimulatory characteristic of hIL-10 has been attributed to a single amino acid, isoleucine at position 87, which in vIL-10 is alanine. A mutant hIL-10 in which isoleucine has been substituted (mut.hIL-10) is biologically active with only immunosuppressive, but not immunostimulatory, functions, making it a potentially superior therapeutic for inflammatory diseases. To compare the efficacy of mut.hIL-10 with hIL-10 and vIL-10 in blocking the progression of rheumatoid arthritis, we used replication defective adenoviral vectors to deliver intra-articularly the gene encoding hIL-10, vIL-10 or mut.hIL-10 to antigen-induced arthritic (AIA) knee joints in rabbits. Intra-articular expression of hIL-10, vIL-10, and mut.hIL-10 resulted in significant improvement of the pathology in the treated joints to similar levels. These observed changes included a significant reduction in intra-articular leukocytosis and the degree of synovitis, as well as normalization of cartilage matrix metabolism. Our results suggest that hIL-10, vIL-10, and mut.hIL-10 are all equally therapeutic in the rabbit AIA model for treating disease pathology.

Adenoviridae↗
PubMed2006

Widespread and stable pancreatic gene transfer by adeno-associated virus vectors via different routes.

Diabetes is a disease of epidemic proportions and is on the rise worldwide. Gene therapy has been actively pursued but limited by technical hurdles and profound inefficiency of direct gene transfer to the pancreas in vivo. Here, we show that, for the first time, appropriate serotypes of adeno-associated virus (AAV), coupled with a double-stranded vector DNA cassette, enable extensive and long-term in vivo gene transfer in the adult mouse pancreas by three different delivery methods. Intraperitoneal and intravenous delivery of AAV8 effectively transduced exocrine acinar cells as well as endocrine beta-cells, while local pancreatic intraductal delivery of AAV6 showed the best efficiency in the beta-cells among all AAV serotypes tested in this study. Nearly the entire islet population showed gene transfer but with distinct gene transfer efficiency and patterns when different delivery methods and vectors were used. Importantly, localized gene delivery coupled with an insulin promoter allowed extensive yet specific gene expression in the beta-cells. These effective new methods should provide useful tools to study diabetes pathogenesis and gene therapy.

Actins↗
PubMed2005

Exosomes derived from genetically modified DC expressing FasL are anti-inflammatory and immunosuppressive.

We previously have demonstrated the ability of primary murine bone marrow-derived DC (BM-DC), genetically modified by adenoviral infection to express FasL, to inhibit progression of established collagen-induced arthritis (CIA) following systemic delivery. Here we demonstrate that exosomes derived from genetically modified BM-DC expressing FasL are able to inhibit inflammation in a murine footpad model of delayed-type hypersensitivity (DTH). Local administration of exosomes derived from DC expressing FasL (Exo/FasL) as well as the parental DC/FasL resulted in a significant reduction in swelling in both the treated and the untreated distal paw. However, both the DC/FasL and the Exo/FasL were unable to suppress the DTH response in lpr (Fas-deficient) mice. Gene transfer of FasL to BM-DC from gld (FasL-deficient) mice resulted in restoration of the ability of DC as well as DC-derived exosomes to suppress DTH. The ability of DC-derived exosomes and DC to suppress DTH responses was antigen specific and MHC class II dependent, but class I independent. The injected exosomes were found to be internalized into CD11c(+) cells at the site of injection and in the draining popliteal lymph node. Systemic injection of exosome/FasL into mice with established CIA resulted in significant disease amelioration. These results demonstrate that both systemic and local administration of exosomes derived from FasL-expressing DC are able to suppress antigen-specific immune responses through an MHC class II-dependent pathway, resulting in effective and sustained treatment of established collagen-induced arthritis and suppression of the DTH inflammatory response. These results suggest that DC/FasL-derived exosomes could be used clinically for the treatment of inflammatory and autoimmune diseases.

Animals↗
PubMed2005

The 3rd International Meeting on Gene Therapy in Rheumatology and Orthopaedics.

The 3rd International Meeting on Gene Therapy in Rheumatology and Orthopaedics was held in Boston, Massachusetts, USA in May 2004. Keystone lectures delivered by Drs Joseph Glorioso and Inder Verma provided comprehensive, up-to-date information on all major virus vectors. Other invited speakers covered the application of gene therapy to treatment of arthritis, including the latest clinical trial in rheumatoid arthritis, as well as lupus and Sjögren's syndrome. Applications in mesenchymal stem cell biology, tissue repair, and regenerative medicine were also addressed. The field has advanced considerably since the previous meeting in this series, and further clinical trials seem likely.

Genetic Therapy↗
PubMed2005

Gene transfer to human joints: progress toward a gene therapy of arthritis.

This article describes the clinical application of gene therapy to a nonlethal disease, rheumatoid arthritis (RA). Intraarticular transfer of IL-1 receptor antagonist (IL-1Ra) cDNA reduces disease in animal models of RA. Whether this procedure is safe and feasible in humans was addressed in a phase I clinical study involving nine postmenopausal women with advanced RA who required unilateral sialastic implant arthroplasty of the 2nd-5th metacarpophalangeal (MCP) joints. Cultures of autologous synovial fibroblasts were established and divided into two. One was transduced with a retrovirus carrying IL-1Ra cDNA; the other provided untransduced, control cells. In a dose escalation, double-blinded fashion, two MCP joints were injected with transduced cells, and two MCP joints received control cells. One week later, injected joints were resected and examined for evidence of successful gene transfer and expression by using RT-PCR, ex vivo production of IL-1Ra, in situ hybridization, and immunohistochemistry. All subjects tolerated the protocol well, without adverse events. Unlike control joints, those receiving transduced cells gave positive RT-PCR signals. Synovia that were recovered from the MCP joints of intermediate and high dose subjects produced elevated amounts of IL-1Ra (P = 0.01). Clusters of cells expressing high levels of IL-1Ra were present on synovia of transduced joints. No adverse events occurred. Thus, it is possible to transfer a potentially therapeutic gene safely to human rheumatoid joints and to obtain intraarticular, transgene expression. This conclusion justifies additional efficacy studies and encourages further development of genetic approaches to the treatment of arthritis and related disorders.

Aged↗
PubMed2005

Exosomes derived from IL-10-treated dendritic cells can suppress inflammation and collagen-induced arthritis.

We have demonstrated previously that local, adenoviral-mediated gene transfer of viral IL-10 to a single joint of rabbits and mice with experimental arthritis can suppress disease in both the treated and untreated contralateral joints. This contralateral effect is mediated in part by APCs able to traffic from the treated joint to lymph nodes as well as to untreated joints. Moreover, injection of dendritic cells (DC) genetically modified to express IL-4 or Fas ligand was able to reverse established murine arthritis. To examine the ability of exosomes derived from immunosuppressive DCs to reduce inflammation and autoimmunity, murine models of delayed-type hypersensitivity and collagen-induced arthritis were used. In this study, we demonstrate that periarticular administration of exosomes purified from either bone marrow-derived DCs transduced ex vivo with an adenovirus expressing viral IL-10 or bone marrow-derived DCs treated with recombinant murine IL-10 were able to suppress delayed-type hypersensitivity responses within injected and untreated contralateral joints. In addition, the systemic injection of IL-10-treated DC-derived exosomes was able suppress the onset of murine collagen-induced arthritis as well as reduce severity of established arthritis. Taken together, these data suggest that immature DCs are able to secrete exosomes that are involved in the suppression of inflammatory and autoimmune responses. Thus DC-derived exosomes may represent a novel, cell-free therapy for the treatment of autoimmune diseases.

Animals↗
PubMed2005

The adeno associated viral vector as a strategy for intradiscal gene transfer in immune competent and pre-exposed rabbits.

STUDY DESIGN: Experimental animal study. OBJECTIVES: This study evaluates the in vitro and in vivo transduction efficacy and transgene expression in immune competent and pre-exposed rabbits. SUMMARY OF BACKGROUND DATA: Degenerative disc disease (DDD) continues to pose a substantial clinical problem. Therapeutic options such as an interbody fusion are highly invasive and result in the loss of the intervertebral disc. In addition, interbody fusion puts the adjacent discs at an even higher risk for disc degeneration. A novel approach to slow DDD is to introduce high levels of growth factors into the degenerating disc by delivering the gene coding for the appropriate growth factor. The most efficient technique to do so to date uses viral vectors. However, viral vectors may be problematic because of their immunogenicity. The adeno-associated virus (AAV) viral vector is known to be less immunogenic than commonly used adenoviral vectors. METHODS: Human nucleus pulposus cells were transduced in vitro. Twenty-four Rabbits were injected with AAV viral vectors carrying different marker genes. Transgene expression and the humoral/cellular immune response to the vector was evaluated. RESULTS: We could show that the AAV viral vector transduces human as well as rabbit nucleus pulposus cells in vitro and in vivo. There is a significant humoral immune response against the AAV vector that decreases transgene expression over 10-fold in preimmunized animals. CONCLUSIONS: AAV is a valuable new vector to achieve transgene expression in the intervertebral disc. In preimmunized animals, its use needs to be further evaluated because of the significant reduction in transgene expression.

Animals↗