Multiparameter Telemetry as a Sensitive Screening Method to Detect Vaccine Reactogenicity in Mice

Refined vaccines and adjuvants are urgently needed to advance immunization against global infectious challenges such as HIV, hepatitis C, tuberculosis and malaria. Large-scale screening efforts are ongoing to identify adjuvants with improved efficacy profiles. Reactogenicity often represents a major hurdle to the clinical use of new substances. Yet, irrespective of its importance, this parameter has remained difficult to screen for, owing to a lack of sensitive small animal models with a capacity for high throughput testing. Here we report that continuous telemetric measurements of heart rate, heart rate variability, body core temperature and locomotor activity in laboratory mice readily unmasked systemic side-effects of vaccination, which went undetected by conventional observational assessment and clinical scoring. Even minor aberrations in homeostasis were readily detected, ranging from sympathetic activation over transient pyrogenic effects to reduced physical activity and apathy. Results in real-time combined with the potential of scalability and partial automation in the industrial context suggest multiparameter telemetry in laboratory mice as a first-line screen for vaccine reactogenicity. This may accelerate vaccine discovery in general and may further the success of vaccines in combating infectious disease and cancer

Sub-genic intolerance, ClinVar, and the epilepsies: A whole-exome sequencing study of 29,165 individuals

Both mild and severe epilepsies are influenced by variants in the same genes, yet an explanation for the resulting phenotypic variation is unknown. As part of the ongoing Epi25 Collaboration, we performed a whole-exome sequencing analysis of 13,487 epilepsy-affected individuals and 15,678 control individuals. While prior Epi25 studies focused on gene-based collapsing analyses, we asked how the pattern of variation within genes differs by epilepsy type. Specifically, we compared the genetic architectures of severe developmental and epileptic encephalopathies (DEEs) and two generally less severe epilepsies, genetic generalized epilepsy and non-acquired focal epilepsy (NAFE). Our gene-based rare variant collapsing analysis used geographic ancestry-based clustering that included broader ancestries than previously possible and revealed novel associations. Using the missense intolerance ratio (MTR), we found that variants in DEE-affected individuals are in significantly more intolerant genic sub-regions than those in NAFE-affected individuals. Only previously reported pathogenic variants absent in available genomic datasets showed a significant burden in epilepsy-affected individuals compared with control individuals, and the ultra-rare pathogenic variants associated with DEE were located in more intolerant genic sub-regions than variants associated with non-DEE epilepsies. MTR filtering improved the yield of ultra-rare pathogenic variants in affected individuals compared with control individuals. Finally, analysis of variants in genes without a disease association revealed a significant burden of loss-of-function variants in the genes most intolerant to such variation, indicating additional epilepsy-risk genes yet to be discovered. Taken together, our study suggests that genic and sub-genic intolerance are critical characteristics for interpreting the effects of variation in genes that influence epilepsy

Endothelial cell seeding improves patency of synthetic vascular grafts: manual versus automatized method

Lack of an endothelial surface is the most important variable causing the relatively poor patency of synthetic bypass grafts. This study was designed to investigate the effect of endothelial cell seeding on small-diameter Dacron grafts seeded with microvascular endothelial cells from omentum, and to evaluate two methods (manual vs automatized) for one-stage seeding in a canine carotid artery model. In 30 mongrel dogs microvascular endothelial cells were harvested from omentum, either by a manual or an automatized method, and seeded onto 6-mm internal diameter Dacron prostheses prior to the graft interposition into the common carotid arteries. Non-seeded Dacron grafts were used as control grafts. All dogs received dipyridamole (75 mg/day) and acetylsalicylic acid (325 mg/day) for 4 weeks. The prostheses were explanted between 2 and 26 weeks after insertion. The results were assessed by patency, angiography, light and scanning electron microscopy, transmission electron microscopy, and morphometry. Endothelial cell seeding improved the patency rate significantly, regardless of the seeding methods used. The overall actuarial patency rates at 5, 12, and 26 weeks were 98%, 94% and 94%, respectively, for the seeded Dacron grafts, and 92%, 62% and 54%, respectively, for the non-seeded grafts. The automatized method yielded more endothelial cells per gram of omental tissue than the manual method (P = 0.0002), but there was no difference (P = 0.34) between the seeding densities per square centimeter of the graft surface. The harvesting and seeding by the automatized method took 55 min for the whole procedure, 20 min less than the manual method. We concluded that one-stage endothelial cell seeding with omental microvascular endothelial cells improved the patency of small-diameter Dacron grafts in a canine model. The automatized method obtained excellent results comparable to the manual procedure, and also reduced the time necessary for the cell seeding

Langzeitresultate in der Hundekarotis mit kleinlumigen Gefassprothesen mit mikrovaskularen Endothelzellen. [Long-term results in the dog carotid artery with small lumen vascular prostheses with microvascular endothelial cells]

In this study we evaluated the long-term results of microvascular endothelial cell seeding of small diameter Dacron grafts with omentally derived cells in a canine model. 6 cm long and 4 mm I.D. seeded Dacron prostheses were implanted end-to-end in the carotid position in 12 dogs for 6 to 12 months. Microvascular endothelial cells were enzymatically harvested from omentum prior to implantation and seeded onto Dacron grafts with a seeding density of 1.5 x 10(6) cells/cm2 of the graft. The antiplatelet therapy (Aspirin, Dipyridamol) was administered for 4 weeks postoperatively. All seeded grafts were patent throughout the study. The thrombus-free surface area for seeded grafts was 99.6 +/- 0.8% and 99.6 +/- 0.9% at 6 months and one year, respectively. Scanning electron microscopy revealed a confluent endothelial layer. We concluded that endothelial cell seeding of smaller-diameter prosthetic vascular grafts with omentally derived endothelial cells obtained excellent long-term patency rate in the canine model

Immediate shear stress resistance of endothelial cell monolayers seeded in vitro on fibrin glue-coated ePTFE prostheses

The shear stress resistance of endothelial cells (EC) previously seeded onto ePTFE grafts was assessed by morphometric determination of the number of cells per cm2 of graft surface before and after exposure of 6 h of arterial blood flow interposed in the canine femoral artery. Autologous venous endothelial cells (AVEC) were harvested from the extrajugular veins of five dogs. The AVEC were cultured in vitro and seeded at a density of 150 x 10(3) cells per cm2 onto 4 mm ID ePTFE grafts precoated with fibrin glue and human fibronectin. Subsequently, the AVEC monolayers on the grafts were cultured for 8 days using a perfusion system and then implanted end-to-end in the femoral artery. All grafts remained patent (5/5). Scanning electron microscopy demonstrated complete, thrombus-free monolayers of AVEC after 6 h of arterial blood flow. The cell densities were 124 +/- 14 and 129 +/- 7 x 10(3) cells per cm2 respectively before and after implantation. It is concluded that in vitro lining of 4 mm ePTFE vascular prostheses is feasible and results in EC monolayers on the graft surface which are shear stress resistant and athrombogenic

Superior late patency of small-diameter Dacron grafts seeded with omental microvascular cells: an experimental study

The purpose of the study was to investigate the effect of omental microvascular cell seeding on the patency of small-diameter Dacron prostheses usable for coronary artery bypass grafting. In a canine carotid artery model, each dog (n = 64) received one seeded and one similar nonseeded Dacron prosthesis (internal diameter = 4 or 6 mm). Enzymatically harvested omental microvascular cells (omentum = 27.6 +/- 5.9 g [+/- the standard deviation]; range, 17 to 50 g) were seeded prior to implantation. The seeding density was 1.91 +/- 0.26 [+/- the standard error] x 10(6) cells/cm2 of graft surface. Dipyridamole (75 mg/d) and acetylsalicylic acid (325 mg/d) were administered orally for 4 weeks postoperatively. The prostheses were explanted between 2 and 52 weeks after placement. The results were assessed by angiography; light, scanning electron, and transmission electron microscopy; and morphometry. The seeded grafts developed a uniform luminal monolayer of endothelial cells with minimal platelet or cellular deposition. These grafts also had a significantly higher overall patency rate and significantly larger thrombus-free surface areas than the nonseeded grafts. The overall actuarial patency rates at 1 week, 5, 12, 26, and 52 weeks were 100%, 98%, 93%, 93%, and 93%, respectively, for seeded Dacron grafts and 100%, 91%, 61%, 54%, and 18%, respectively, for nonseeded grafts. The patency rates of Dacron grafts usable for coronary artery bypass grafting are significantly improved by seeding with omental microvascular cells in a canine model

Development of a small-lumen vascular prosthesis coated with autologous endothelial cells

A new compliant prosthesis with a monolayer of autologous endothelial cells (ENC) has been developed. It consists of a porous polyurethane-siloxane-copolymer reinforced by a polyester network to prevent excessive dilatation. On the inner surface an ENC monolayer is established before implantation by a cell culture procedure. The prosthesis displays compliance (13.2 +/- 3.0 x 10(-4) mmHg-1) comparable to native arteries. It is non-kinkable (minimal radius of curvature less than 5 mm). Burst resistance, remaining deformation, cut out force and tensile strength are superior to standard values. ENC-coverage in excess of 95% of the inner surface was produced in vitro using a lining procedure. The monolayer of confluent cells was demonstrated to consist of endothelial cells by their characteristic cobblestone morphology, the expression of factor VIII related antigen and the specific uptake of Dil-Ac-LDL. The unstimulated prostacyclin production was similar both in native veins as well as in lined prostheses. Antithrombogenicity of the endothelial cell lining was demonstrated in 24 h animal implants

Einfluss der hamodynamischen Bedingungen und der Prothesenstruktur auf die Endothelialisierung von klinischen und experimentellen kleinlumigen Gefassprothesen [Effect of hemodynamic conditions and prosthesis structure on endothelialization of clinical and experimental small lumen vascular prostheses]

Seeding of small-diameter vascular prostheses (ID less than or equal to 6 mm) with autologous microvascular cells (AMVC) results in a complete endothelial cell layer on the luminal surface. The purpose of this study was to examine the influence of the blood flow velocity (due to 4 or 6 mm ID) and the structure of inner graft surface (crimped, uncrimped) on the endothelialization. AMVC were harvested from omental adipose tissue (mean: 0.56 X 10(6) cells/g tissue) from 10 mongrel dogs (mean: 27.9 kg). During preclotting, the 4 mm uncrimped and the 6 mm crimped double velour Dacron prostheses (Meadox Medicals, Inc.) were seeded with 1.0 X 10(6) cells/cm2 graft surface. Grafts were implanted into the carotid arteries (N = 5 in each group). The animals received antiplatelet therapy. After five weeks, all seeded prostheses were patent. The thrombus free surface (TFS) of seeded prostheses was 99.9% (4 mm) and 90.5% (6 mm). Scanning electron microscopy revealed an athrombogenic layer of endothelial cells on a smooth surface. -It is concluded that in canine experiments endothelialization of 4 and 6 mm grafts after seeding with AMVC is not affected by blood flow velocity or graft structure

Endothelialisierung von 6-mm-Gefassprothesen mit mikrovaskularen Endothelzellen aus dem Fettgewebe des Omentums. [Endothelialization of 6 mm vascular prostheses with microvascular endothelial cells from fatty tissue of the omentum]

The seeding of 6 mm Dacron prostheses with microvascular endothelial cells from omental adipose tissue leads to endothelialized prostheses, 5 weeks later with a complete coverage of functional and thrombus free endothelium. The method is ready for clinical trials

A compliant small-diameter vascular prosthesis lined with functional venous endothelial cells

The long-term patency of small-diameter vascular grafts is still unsatisfactory. In contrast to native arteries, they are inelastic and lack active antithrombogenicity. To improve long-term patency, a new 4 mm internal diameter prosthesis was developed which is compliant and lined with functional endothelial cells (ENC). The wall of this prosthesis consists of a microporous polyurethane-siloxane copolymer reinforced with a polyester network. It displays compliance (13.2 x 10(-4) mmHg-1) comparable to native arteries, is nonkinkable (minimum radius of curvature = 5 mm), burst resistant, and easily suturable. Using a lining procedure, coverage of prostheses by ENC was in excess of 95%. The ENC populations were found to be highly pure (by factor VIII-related antigen, DilAcLDL uptake) and to produce about 0.3 ng prostacyclin per cm2. In vitro tests of shear stress resistance demonstrated that ENC monolayers on the new elastic prosthesis remain intact for 3 hr in physiologically pulsating culture medium (Vmax = 50 cm/sec). Lined prostheses implanted for 24 hours in mongrel dogs as an arteriovenous shunt demonstrated the antithrombogenicity of the cultured ENC. The results suggest that small-diameter vascular prostheses which are compliant, porous, and actively antithrombogenic are feasible