Ad Fibers Research Articles

Fiber manipulation and post-assembly nanobody conjugation for adenoviral vector retargeting through SpyTag-SpyCatcher protein ligation.

For adenoviruses (Ads) to be optimally effective in cancer theranostics, they need to be retargeted toward target cells and lose their natural tropism. Typically, this is accomplished by either engineering fiber proteins and/or employing bispecific adapters, capable of bonding Ad fibers and tumor antigen receptors. This study aimed to present a simple and versatile method for generating Ad-based bionanoparticles specific to target cells, using the SpyTag-SpyCatcher system. The SpyTag peptide was inserted into the HI loop of fiber-knob protein, which could act as a covalent anchoring site for a targeting moiety fused to a truncated SpyCatcher (SpyCatcherΔ) pair. After confirming the presence and functionality of SpyTag on the Ad type-5 (Ad5) fiber knob, an adapter molecule, comprising of SpyCatcherΔ fused to an anti-vascular endothelial growth factor receptor 2 (VEGFR2) nanobody, was recombinantly expressed in Escherichia coli and purified before conjugation to fiber-modified Ad5 (fmAd5). After evaluating fmAd5 detargeting from its primary coxsackie and adenovirus receptor (CAR), the nanobody-decorated fmAd5 could be efficiently retargeted to VEGFR2-expressing 293/KDR and human umbilical vein endothelial (HUVEC) cell lines. In conclusion, a plug-and-play platform was described in this study for detargeting and retargeting Ad5 through the SpyTag-SpyCatcher system, which could be potentially applied to generate tailored bionanoparticles for a broad range of specific targets; therefore, it can be introduced as a promising approach in cancer nanotheranostics.

Effect of different chemical treatments and loadings of Arundo donax L. fibers on the dynamic mechanical, thermal, and morphological properties of bisphenol A aniline based polybenzoxazine composites

AbstractIn this research paper, bisphenol A aniline based benzoxazine (BA‐a) was reinforced with natural, inexpensive, and largely distributed cellulosic plant, namely Arundo donax L. (AD) at various loadings of 5, 15, and 25 wt%. AD fibers were initially subjected to three different chemical surface modifications using alkaline, silane, and combined alkali‐silane solutions. The main purpose of this work was to investigate the dynamic mechanical properties, morphological stability, and thermal stability of the as‐prepared composites by pointing out the effects of natural reinforcements treated in different ways at different loadings. Dynamic mechanical analysis of the prepared composites revealed an enhancement in storage modulus (E′) and loss modulus (E″) to higher values, especially for composites reinforced by alkali‐ or combined‐treated fibers due to the strong effect of alkaline solution to remove noncellulosic matters. Composite loaded at 25% of alkali‐silane treated AD exhibited a glass transition temperature (Tg) of 217°C, while pure polymer had a Tg of 188°C. The enhanced properties are a great indicator of the high fiber–matrix adhesion and compatibility. This conclusion is strengthened by scanning electron microscopy images. The thermal stability and degradation of the different biocomposites are also investigated by thermogravimetric analysis, revealing that the nature of the employed chemical treatment affected considerably the thermal behavior and the char yield of the composites.

Sickle Cell Disease Patients Show Sensitization of Myelinated Sensory Nerve Fibers

There is increasing evidence that many patients with sickle cell disease (SCD) develop chronic pain in addition to experiencing acute pain secondary to vaso-occlusive episodes (VOE). Those patients who develop chronic pain often exhibit features suggestive of neuropathic processes such as allodynia and pain "shooting" or "tingling" in character. Using electrophysiologic techniques in ex-vivo nerve-skin preparations and nocifensive behavior studies in sickle cell mouse models, we and others have shown that SCD is associated with sensitization of light touch cutaneous sensory fibers, and decreased threshold in response to 2000Hz and 250 Hz electrical sine wave stimulation. These findings are indicative of Aβ and Ad fibers sensitization and in turn compatible with neuropathic process.We hypothesized that the same phenomena of nerve fiber sensitization in the mouse model would be observed in SCD patients. This hypothesis was tested using an experimental paradigm with sine-wave electrical stimulation delivered at three different frequencies: 2000, 250, and 5 Hz, which preferentially stimulate Aβ, Ad, and C fibers, respectively. Thermal and mechanical sensory testing was also performed. After IRB approval, SCD subjects with high (≥ than 3 ER visits or admissions for VOEs per year) or low (< 2 ER visits or admissions per year) pain frequency, and age- and sex-matched African-American healthy controls were recruited at baseline states during routine clinic visits. After informed consent (and/or assent) was obtained, patients underwent quantitative sensory testing (QST) that included heat and cold perception and tolerance (with a TSA II-Sensory Analyzer), mechanical (using a Wagner FDIX Force One) and current perception and pain tolerance thresholds (using the Neurometer).We enrolled 19 SCD subjects with high and 4 with low pain frequency (total 23 SCD participants), and 11 African-American non-SCD subjects who tolerated and completed all QSTs. As there was no difference between QST responses in patients with high and low pain frequency, these two groups were combined as the SCD group. We found that SCD subjects had significantly decreased cold tolerance threshold (p=0.034) and a trend towards lower thresholds for cold perception and heat tolerance, which is in concert with previously reported findings. Expanding upon these findings, SCD patients had a significantly reduced pain tolerance threshold in response to 2000 Hz stimulation (p=0.005), which preferentially stimulates Aβ fibers (see figure). These results suggest that adolescents with SCD have sensitization of myelinated Aβ fibers. Interrogation of the Ad and C fibers with 250 and 5 Hz stimulation, respectively, revealed no significant differences in tolerance thresholds among the groups, however SCD patients showed a trend toward lower thresholds in both frequencies (see figure). Combined, these findings support the use of sine-wave stimulation on the evaluation of pain phenotype in SCD and the notion that SCD subjects may have neuropathic processes contributing to their pain phenotype. TableDemographics, thermal and mechanical sensory testing in SCD and control subjects.VariableSCD (n=23)Control (n=11)Age (years)16 (15-18.8)19 (14-21)Male (%)3 (30%)11 (48%)Cold Perception (°C)29.8 (28.5-30.5)30.4 (29.9-30.9)Cold Tolerance (°C)8.5 (2.0-17.0) *0.9 (0.0-6.9)Heat Perception (°C)34.4 (33.5-35.2) *33.6 (33.2-34.1)Heat Tolerance (°C)48.9 (47.5-49.8)50.0 (45.3-50.1)Mechanical Tolerance (lbF)7.7 (5.9-9.7)9.5 (7.7-10.8)*p < 0.05. Values are shown as median and interquartile range. SCD subjects reached cold tolerance and heat perception at significantly higher temperatures as compared to controls; p=0.034 and p= 0.035, respectively. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Neural electrophysiological features of laser-evoked potentials and their applications

Pain is related to the encoding and processing of afferent nociceptive information in the brain, and is a critical factor that allows humans and animals to learn to avoid potential or actual tissue injury. Being conducted by Ad fibers and C fibers, nociceptive signals are processed by multiple brain structures, and normally generate a double sensation: an initial pricking pain (first pain) and a prolonged burning sensation (second pain). Intense laser heat pulses can selectively excite nociceptive free nerve endings in superficial layers of the skin. Such stimuli can elicit changes in brain responses (laser-evoked potentials, LEPs) in the ongoing electroencephalograms (EEGs). Until now, six reliable components have been detected in Ad-LEPs: Ad-N1, Ad-N2, Ad-P2, and Ad-P4 waves in the time domain and alpha-band event-related desynchronization (α-ERD) and gamma-band event-related synchronization (γ-ERS) in the time- frequency domain. Meanwhile, by optimizing the experimental paradigm and parameters, two components of C-LEPs have been reliably detected: C-N2 and C-P2 waves in the time domain. The present paper reviewed the neural electrophysiological features of LEPs and their cognitive significance. LEPs can not only be used to investigate the peripheral and central processing of nociceptive sensory input, which is important in several clinical applications, but also be adopted in some basic research (e.g., the aging problem and social cognition) as a novel neurophysiological technique.

MP1-09 ASSOCIATION BETWEEN AGE-RELATED DETRUSOR UNDERACTIVITY AND DOWNREGULATION OF THE BIOLOGICAL CLOCK OF CONNEXIN 43-DERIVED GAP JUNCTIONS IN RAT BLADDERS

INTRODUCTION AND OBJECTIVES: Various diseases can cause altered afferent autonomic sensitivity of bladder, including diabetes, neurologic or vascular lesions, multiple sclerosis, recurrent infection, interstitial cystitis, pelvic pain syndrome. In current urologic clinical practice, to study autonomic and sensory bladder neuropathy, clinicians still rely on patientsi?/2 self-reported bladder sensation during the filling phase of urodynamics. We developed and tested the efficacy of an implantable bladder device which, when combined with the Neurometeri?/2, can be used to assess fiber specific afferent bladder sensation in the mouse. METHODS: We developed an implantable bladder device that applies selective nerve fiber stimuli (250 Hz for small myelinated Ad fibers and 5 Hz for unmyelinated C fibers) to the bladder mucosa in the mouse to determine bladder current perception threshold (CPT) values. We performed 4 experiments in 64 C57BL/6J mice to examine the effects of our device on voiding habits, assess the interobserver reliability of the sensory perception threshold and determine the effects of intravesical administration of resiniferatoxin and lidocaine on the sensory perception threshold. RESULTS: Sensory perception threshold values obtained by 2 blinded, independent observers were not different from each other (p1⁄40.74382). CPT values obtained at the 2 stimulation frequencies remained constant for at least 6 days after device implantation. A significant increase in CPT values after resiniferatoxin instillation was noted at a stimulus frequency of 5 Hz (p1⁄40.01347), whereas intravesical lidocaine led to an immediate increase in CPT at 250 (p1⁄40.0003) and 5 Hz (p1⁄40.0001). The implantation led to an early decreased voided volume and increased frequency of voids at 4 days, although these parameters returned to close normal after 6 days. The implanted bladder histology reveals defect created by device and mild inflammatory changes in next areas. CONCLUSIONS: Assessment of bladder afferent sensation with our newly developed device is feasible in mice. It provides CPTs that appear to be fiber-type selective for autonomic bladder afferent nerves.

Anaerobic treatment of lignocellulosic material to co-produce methane and digested fiber for ethanol biorefining

Five different ratios of corn stover to swine manure were investigated to evaluate the performance of anaerobic digestion and the quality of anaerobically digested fiber (AD fiber) as a feedstock for bioethanol production. The stover-to-manure ratio of 40:60 generated 364L biogas and 797g AD fiber per kg of dry raw feedstock daily. The AD fibers after digestion were pretreated and hydrolyzed to release sugars for ethanol fermentation. The stover-to-manure ratio of 40:60 was able to produce 152g methane and 50g ethanol per kg of dry raw feedstock. The net energy generated from the ratio 40:60 was 5.5MJkg−1 dry raw feed, which was an 18% increase on net energy output compared to the other ratios and proved to be most beneficial for a biorefinery.

Development of a new bioethanol feedstock – Anaerobically digested fiber from confined dairy operations using different digestion configurations

Abstract Two types of digesters, continuous stirring-tank reactor (CSTR) and plug flow reactor (PFR), were integrated into a biorefining concept to generate a new cellulosic ethanol feedstock –anaerobically digested fiber (AD fiber) from dairy cow feces. Cellulose content in AD fibers was significantly increased during the anaerobic digestion. CSTR and PFR AD fibers had cellulose contents of 357 and 322 g kg −1 dried AD fiber. The AD fibers were enzymatically hydrolyzed after being pretreated by dilute sulfuric acid or dilute sodium hydroxide, and the hydrolysates were used to produce ethanol. Alkali pretreatment was concluded as a suitable pretreatment method for AD fibers. Under the optimal conditions the AD fibers processed by CSTR and PFR produced ethanol of 26 g kg −1 and 23 g kg −1 dry feces, respectively. Energy balance analysis further indicated that CSTR was a preferred digestion method to prepare AD fiber for ethanol production.

Structural variations in species B adenovirus fibers impact CD46 association.

A majority of species B adenoviruses (Ads) use CD46 as their primary receptor; however, the precise mechanisms involved in the binding of different Ad types to CD46 have not been resolved. Although previous studies indicate close similarities between two members of species B2 Ads in their usage of CD46, our current investigations revealed a surprisingly low CD46 binding affinity of the species B1 Ad16 fiber knob (equilibrium dissociation constant of 437 nM). We determined the crystal structure of the Ad16 fiber knob and constructed a model of this protein in complex with CD46. A comparison of this model to that of the CD46-Ad11 complex revealed structural differences in the FG and IJ loops that are part of the CD46 binding site. An analysis of a panel of recombinant fiber knobs with mutations targeting these regions in Ad16 and Ad11 uncovered a major contribution of the FG loop on CD46 binding. Two extra residues in the FG loop of the Ad16 fiber significantly reduce receptor interaction. Although avidity effects permit the use of CD46 on host cells by Ad16, virus binding occurs with lower efficiency than with B2 Ad types. The longer FG loop of the Ad16 fiber knob also is shared by other species B1 Ad fibers and, thus, may contribute to the low CD46 binding efficiencies observed for these Ad types. Our findings provide a better understanding of how different Ad types associate with CD46 and could aid in the selection of specific Ad fibers for more efficient Ad gene delivery vectors.

Conservation of fiber structure and CD46 usage by subgroup B2 adenoviruses

Most subgroup B2 adenoviruses use CD46 as their primary receptor. Recent structural and mutagenesis studies suggested that Ad11 and Ad35 likely engage this receptor in a very similar fashion. However, no comparative studies assessing the cell-associated CD46 binding efficiencies of different Ad fibers have been performed. We solved the crystal structure of Ad35 fiber knob and constructed a model of the fiber knob complexed with CD46. Comparison of our model with that of Ad11–CD46 showed that despite a larger CD46-interacting region in the IJ loop of Ad11, the buried surface area was very similar, suggesting that both fiber knobs might exhibit similar binding. In support of this, cell based competition studies demonstrated almost identical binding efficiencies of Ad11 and Ad35 fibers to cell surface CD46. These findings shed further light on CD46 association by Ad and could impact the selection of novel Ad types for gene transfer.

Development of Renal-targeted Vectors Through Combined In Vivo Phage Display and Capsid Engineering of Adenoviral Fibers From Serotype 19p

The potential efficacy of gene delivery is dictated by the infectivity profile of existing vectors, which is often restrictive. In order to target cells and organs for which no efficient vector is currently available, a promising approach would be to engineer vectors with novel transduction profiles. Applications that involve injecting adenovirus (Ad) vectors into the bloodstream require that native tropism for the liver be removed, and that targeting moieties be engineered into the capsid. We previously reported that pseudotyping the Ad serotype 5 fiber for that of Ad19p results in reduced hepatic transduction. In this study we show that this may be caused, at least in part, by a reduction in the capacity of the Ad19p-based virus to bind blood coagulation factors. It is therefore a potential candidate for vector retargeting, focusing on the kidney as a therapeutic target. We used in vivo phage display in rats, and identified peptides HTTHREP and HITSLLS that homed to the kidneys following intravenous injection. We engineered the HI loop of Ad19p to accommodate peptide insertions and clones. Intravenous delivery of each peptide-modified virus resulted in selective renal targeting, with HTTHREP and HITSLLS-targeted viruses selectively transducing tubular epithelium and glomeruli, respectively. Our study has important implications for the use of genetic engineering of Ad fibers to produce targeted gene delivery vectors.

Evaluation of Biodistribution and Safety of Adenovirus Vectors Containing Group B Fibers After Intravenous Injection into Baboons

Vectors containing group B adenovirus (Ad) fibers are able to efficiently transduce gene therapy targets that are refractory to infection with standard Ad serotype 5 (Ad5) vectors, including malignant tumor cells, hematopoietic stem cells, and dendritic cells. Preliminary studies in mice indicate that, after intravenous injection, B-group fiber-containing Ads do not efficiently transduce most organs and cause less acute toxicity than Ad5 vectors. However, biodistribution and safety studies in mice are of limited value because the mouse analog of the B-group Ad receptor, CD46, is expressed only in the testis, whereas in humans, CD46 is expressed on all nucleated cells. Unlike mice, baboons have CD46 expression patterns and levels that closely mimic those in humans. We conducted a biodistribution and toxicity study of group B Ad fiber-containing vectors in baboons. Animals received phosphate-buffered saline, Ad5-bGal (a first-generation Ad5 vector), or B-group fiber-containing Ads (Ad5/35-bGal and Ad5/11-bGal) at a dose of 2 x 10(12) VP/kg, and vector biodistribution and safety was analyzed over 3 days. The amount of Ad5/35-bGal and Ad5/11-bGal vector genomes was in most tissues one to three orders of magnitude below that of Ad5. Significant Ad5/35- and Ad5/11-mediated transgene (beta-galactosidase) expression was seen only in the marginal zone of splenic follicles. Compared with the animal that received Ad5-bGal, all animals injected with B-group fiber-containing Ad vectors had lower elevations in serum proinflammatory cytokine levels. Gross and histopathology were normal in animals that received B-group Ad fiber-containing Ads, in contrast to the Ad5-infused animal, which showed widespread endothelial damage and inflammation. In a further study, a chimeric Ad5/35 vector carrying proapoptotic TRAIL and Ad E1A genes under tumor-specific regulation was well tolerated in a 30-day toxicity study. No major clinical, serologic, or pathologic abnormalities were noticed in this animal.

138. Blood Factors Affect Adenovirus Infectivity and Bio-Distribution In Vivo

An incomplete understanding of adenovirus (Ad) interactions with host factors has significantly hampered application of Ad-based vectors for human gene therapy. This is particularly true following intravascular delivery, when the bulk of the vector dose is rapidly cleared from the blood and transduction of liver tissue and innate immune responses contribute to the overall poor performance of Ad as a therapeutic vector. Accumulating data suggest that liver-mediated Ad clearance from the blood and Ad infection of liver cells do not occur through the classical model of Ad infection, which relies on the presence of the coxsakie- and adenovirus receptor, CAR, and alpha-v integrins. Both, CAR and/or integrin binding-ablated vectors exert dramatic innate immune responses and are rapidly cleared by the liver upon systemic application. Recently, we discovered that blood coagulation factor IX directly binds to the Ad fiber knob domain and mediates hepatocyte and Kupffer cell infection in vivo through interaction with novel hepatocellular receptors. Our data, however, indicate that in addition to FIX, other blood factors are likely to contribute to Ad liver cell infection. To study in a systematic way host factors interacting with Ad upon intravascular application, we incubated freshly purified mouse plasma with recombinant Ad5 or Ad35 fiber knob domains immobilized on agarose beads, and Ad fiber knob-bound proteins were recovered and analyzed by tandem mass spectrometry. These analyses revealed that multiple plasma proteins efficiently bind to the Ad fiber knob domain. We confirmed that most of them (including alpha-2 macroglobulin, fibrinogen, complement C3, and complement C4-binding protein, C4BP) are capable of direct binding to Ad fibers in a slot-blot assay. To investigate whether any of the identified Ad-binding plasma proteins can mediate liver cell infection, we infected human hepatocarcinoma cells with an Ad5F|[ast]| vector, possessing a single point mutation abrogating its binding to CAR. While supplementation of Ad5F|[ast]| virus containing media with alpha-2 macroglobulin, fibrinogen or complement C3 resulted in very low background levels of infection, supplementation of media with C4BP completely restored Ad5F|[ast]| infectivity toward these cells. Additionally, we confirmed that C4BP-mediated infection was dose dependent, mediated by the fiber knob domain, and could be efficiently competed with ligands that bind heparan sulfate proteoglycans and LDL-related receptor protein, LRP. C4BP is one of the most abundant plasma proteins, present at an average concentration of 250 |[mu]|g/ml. Clearly, upon intravascular administration, Ads will be exposed to large amounts of C4BP capable of targeting them to liver cells. While further studies should show whether Ad binding to other blood factors is of any physiological significance, our data indicate that there is a complex network of interactions between Ad and plasma proteins, that contribute to the observed pharmacokinetics, bio-distribution and toxicity after intravascular application. Our study helps better understand Ad-host interactions in vivo, when vector applied systemically. It also provides the basis for developing rational approaches toward construction of safe and efficient Ad vectors for human gene therapy applications.

145. Evaluation of Biodistribution and Safety of Adenovirus Vectors Containing B-Group Fibers after Intravenous Injection into Baboons

Top of pageAbstract Vectors containing group B adenovirus (Ad) fibers have been shown to efficiently transduce gene therapy targets that are refractory to infection with standard Ad serotype 5 (Ad5) vectors, including malignant tumor cells, hematopoietic stem cells, and dendritic cells. Preliminary studies in mice indicate that B-group fiber containing Ads do not efficiently transduce most organs and cause less acute toxicity than Ad5 vectors after intravenous injection. However, biodistribution and safety studies in mice are of limited value because the mouse analogue of the B-group Ad receptor, CD46, is expressed only in the testis, while in humans, CD46 is expressed on all nucleated cells. Unlike mice, baboons have CD46 expression patterns and levels that closely mimic those in humans. We conducted a biodistribution and toxicity study of group B Ad vectors in baboons. Animals received intravenous injection of PBS, a standard Ad5 vector, or chimeric vectors containing B-group Ad11 or Ad35 fibers (Ad5/35 or Ad5/11) at dose of 2|[times]|1012 vp/kg and biodistribution and safety was analyzed over 3 days. Analysis of Ad5/35 and Ad5/11 vector genome distribution by quantitative PCR revealed uptake into most tissues at levels 1-3 orders of magnitude below that of Ad5. Significant Ad5/35 and Ad5/11-mediated transgene (b-galactosidase) expression was only seen in the marginal zone of splenic follicles. Compared to the animal that received the Ad5 vector, all animals injected with B-group fiber containing Ad vectors had lower elevations in serum pro-inflammatory cytokine levels. Gross and histopathology were normal in animals that received B-group Ad fiber containing Ads, in contrast to the Ad5 infused animal which showed widespread endothelial damage and inflammation (which is in agreement with studies performed by others). In a further study, a chimeric Ad5/35 vector carrying pro-apoptotic TRAIL and Ad E1A genes under tumor-specific regulation was well tolerated in a 30 day toxicity study. No major clinical, serologic or pathologic abnormalities were noticed in this animal. Overall, this study suggests that Ad vectors possessing B-group Ad fibers have a better safety profile after intravenous injection than conventional Ad5-based vectors. This makes them potential candidates for gene therapy, for example in treatment of metastatic cancer or cardiovascular diseases.

Development of Efficient Viral Vectors Selective for Vascular Smooth Muscle Cells

The vascular smooth muscle cell (SMC) is integral to the pathogenesis of neointimal formation associated with late vein graft failure, in-stent restenosis, and transplant arteriopathy. Viral vectors transduce SMC with low efficiency and hence, there is a need for improvement. We aimed to enhance the efficiency and selectivity of gene delivery to human SMC. Targeting ligands were identified using phage display on primary human saphenous vein SMC with linear and cyclic libraries. Two linear peptides, EYHHYNK (EYH) and GETRAPL (GET), were incorporated into the HI loop of adenovirus (Ad) fibers and the capsid protein of adeno-associated virus-2 (AAV-2). Exposure of human venous SMC to EYH-modified (but not the GET-modified) Ad vector resulted in a significant increase in transgene expression levels at short, clinically relevant exposure times. Similarly, the EYH-modified AAV vector resulted in enhanced gene transfer to human venous SMC but not endothelial cells in a time- and dose-dependent manner. The EYH-modified AAV vector also enhanced (up to 70-fold) gene delivery to primary human arterial SMC. Hence, incorporation of EYH into Ad and AAV capsids resulted in a significant and selective enhancement in transduction of SMC and has implications for improving local gene delivery to the vasculature.