Allogeneic chimerism with low-dose irradiation, antigen presensitization, and costimulator blockade in H-2 mismatched mice

We have previously shown that the keys to high-level nontoxic chimerism in syngeneic models are stem cell toxic, nonmyelotoxic host treatment as provided by 100-cGy whole-body irradiation and relatively high levels of marrow stem cells. This approach was unsuccessful in H-2 mismatched B6.SJL to BALB/c marrow transplants, but with tolerization, stable multilineage chimerism was obtained. Ten million B6.SJL spleen cells were infused intravenously into BALB/c hosts on day -10 and (MR-1) anti-CD40 ligand monoclonal antibody (mAb) injected intraperitoneally at varying levels on days -10, -7, -3, 0, and +3 and the BALB/c mice irradiated (100 cGy) and infused with 40 million B6.SJL/H-2 mismatched marrow cells on day 0. Stable multilineage chimerism at levels between 30% to 40% was achieved in the great majority of mice at 1.6 mg anti-CD40 ligand mAb per injection out to 64 weeks after transplantation, without graft-versus-host disease. The transplanted mice were also tolerant of donor B6.SJL, but not third-party CBA/J skin grafts at 8 to 9 and 39 to 43 weeks after marrow transplantation. These data provide a unique model for obtaining stable partial chimerism in H-2 mismatched mice, which can be applied to various clinical diseases of man such as sickle cell anemia, thalassemia, and autoimmune disorders

Cytokine-facilitated transduction leads to low-level engraftment in nonablated hosts

Using a murine bone marrow transplantation model, we evaluated the long-term engraftment of retrovirally transduced bone marrow cells in nonmyeloablated hosts. Male bone marrow was stimulated in a cocktail of interleukin-3 (IL-3), IL-6, IL-11, and stem cell factor (SCF) for 48 hours, then cocultured on the retroviral producer line MDR18.1 for an additional 24 hours. Functional transduction of hematopoietic progenitors was detected in vitro by reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of multiple drug resistance 1 (MDR1) mRNA from high proliferative potential-colony forming cell (HPP-CFC) colonies. After retroviral transduction, male bone marrow cells were injected into nonablated female mice. Transplant recipients received three TAXOL (Bristol-Myers, Princeton, NJ) injections (10 mg/kg) over a 14-month period. Transplant recipient tissues were analyzed by Southern blot and fluorescence in situ hybridization for Y-chromosome-specific sequences and showed donor cell engraftment of approximately 9%. However, polymerase chain reaction amplification of DNAs from bone marrow, spleen, and peripheral blood showed no evidence of the transduced MDR1 gene. RT-PCR analysis of total bone marrow RNA showed that transcripts from the MDR1 gene were present in a fraction of the engrafted donor cells. These data show functional transfer of the MDR1 gene into nonmyeloablated murine hosts. However, the high rates of in vitro transduction into HPP-CFC, coupled with the low in vivo engraftment rate of donor cells containing the MDR1 gene, suggest that the majority of stem cells that incorporated the retroviral construct did not stably engraft in the host. Based on additional studies that indicate that ex vivo culture of bone marrow induces an engraftment defect concomitantly with progression of cells through S phase, we propose that the cell cycle transit required for proviral integration reduces or impairs the ability of transduced cells to stably engraft

Ultrasound capsule endoscopy:sounding out the future

Video capsule endoscopy (VCE) has been of immense benefit in the diagnosis and management of gastrointestinal (GI) disorders since its introduction in 2001. However, it suffers from a number of well recognized deficiencies. Amongst these is the limited capability of white light imaging, which is restricted to analysis of the mucosal surface. Current capsule endoscopes are dependent on visual manifestation of disease and limited in regards to transmural imaging and detection of deeper pathology. Ultrasound capsule endoscopy (USCE) has the potential to overcome surface only imaging and provide transmural scans of the GI tract. The integration of high frequency microultrasound (µUS) into capsule endoscopy would allow high resolution transmural images and provide a means of both qualitative and quantitative assessment of the bowel wall. Quantitative ultrasound (QUS) can provide data in an objective and measurable manner, potentially reducing lengthy interpretation times by incorporation into an automated diagnostic process. The research described here is focused on the development of USCE and other complementary diagnostic and therapeutic modalities. Presently investigations have entered a preclinical phase with laboratory investigations running concurrently

Ultrasound mediated delivery of quantum dots from a capsule endoscope to the gastrointestinal wall

Biologic drugs, defined as therapeutic agents produced from or containing components of a living organism, are of growing importance to the pharmaceutical industry. Though oral delivery of medicine is convenient, biologics require invasive injections because of their poor bioavailability via oral routes. Delivery of biologics to the small intestine using electronic delivery with devices that are similar to capsule endoscopes is a promising means of overcoming this limitation and does not require reformulation of the therapeutic agent. The efficacy of such capsule devices for drug delivery could be further improved by increasing the permeability of the intestinal tract lining with an integrated ultrasound transducer to increase uptake. This paper describes a novel proof of concept capsule device capable of electronic application of focused ultrasound and delivery of therapeutic agents. Fluorescent markers, which were chosen as a model drug, were used to demonstrate in-vivo delivery in the porcine small intestine with this capsule. We show that the fluorescent markers can penetrate the mucus layer of the small intestine at low acoustic powers when combining microbubbles with focussed ultrasound. These findings suggest that the use of focused ultrasound together with microbubbles could play a role in the oral delivery of biologic therapeutics

Ultrasound mediated delivery of quantum dots from a proof of concept capsule endoscope to the gastrointestinal wall

Biologic drugs, defined as therapeutic agents produced from or containing components of a living organism, are of growing importance to the pharmaceutical industry. Though oral delivery of medicine is convenient, biologics require invasive injections because of their poor bioavailability via oral routes. Delivery of biologics to the small intestine using electronic delivery with devices that are similar to capsule endoscopes is a promising means of overcoming this limitation and does not require reformulation of the therapeutic agent. The efficacy of such capsule devices for drug delivery could be further improved by increasing the permeability of the intestinal tract lining with an integrated ultrasound transducer to increase uptake. This paper describes a novel proof of concept capsule device capable of electronic application of focused ultrasound and delivery of therapeutic agents. Fluorescent markers, which were chosen as a model drug, were used to demonstrate in vivo delivery in the porcine small intestine with this capsule. We show that the fluorescent markers can penetrate the mucus layer of the small intestine at low acoustic powers when combining microbubbles with focused ultrasound during in vivo experiments using porcine models. This study illustrates how such a device could be potentially used for gastrointestinal drug delivery and the challenges to be overcome before focused ultrasound and microbubbles could be used with this device for the oral delivery of biologic therapeutics

The Development of Ovine Gastric and Intestinal Organoids for Studying Ruminant Host-Pathogen Interactions

Gastrointestinal (GI) infections in sheep have significant implications for animal health, welfare and productivity, as well as being a source of zoonotic pathogens. Interactions between pathogens and epithelial cells at the mucosal surface play a key role in determining the outcome of GI infections; however, the inaccessibility of the GI tract in vivo significantly limits the ability to study such interactions in detail. We therefore developed ovine epithelial organoids representing physiologically important gastric and intestinal sites of infection, specifically the abomasum (analogous to the stomach in monogastrics) and ileum. We show that both abomasal and ileal organoids form self-organising three-dimensional structures with a single epithelial layer and a central lumen that are stable in culture over serial passage. We performed RNA-seq analysis on abomasal and ileal tissue from multiple animals and on organoids across multiple passages and show the transcript profile of both abomasal and ileal organoids cultured under identical conditions are reflective of the tissue from which they were derived and that the transcript profile in organoids is stable over at least five serial passages. In addition, we demonstrate that the organoids can be successfully cryopreserved and resuscitated, allowing long-term storage of organoid lines, thereby reducing the number of animals required as a source of tissue. We also report the first published observations of a helminth infecting gastric and intestinal organoids by challenge with the sheep parasitic nematode Teladorsagia circumcincta, demonstrating the utility of these organoids for pathogen co-culture experiments. Finally, the polarity in the abomasal and ileal organoids can be inverted to make the apical surface directly accessible to pathogens or their products, here shown by infection of apical-out organoids with the zoonotic enteric bacterial pathogen Salmonella enterica serovar Typhimurium. In summary, we report a simple and reliable in vitro culture system for generation and maintenance of small ruminant intestinal and gastric organoids. In line with 3Rs principals, use of such organoids will reduce and replace animals in host-pathogen research

Relativistic theories of interacting fields and fluids

We investigate divergence-type theories (DTT) describing the dissipative interaction between a field and a fluid. We look for theories which, under equilibrium conditions, reduce to the theory of a Klein-Gordon scalar field and a perfect fluid. We show that the requirements of causality and positivity of entropy production put non-trivial constarints to the structure of the interaction terms. These theories provide a basis for the phenomonological study of the reheating period.Comment: 17 pages, no figures, minor corrections mad

Bacterial Filtration Using Carbon Nanotube/Antibiotic Buckypaper Membranes

The preparation of free-standing carbon nanotube “buckypaper” (BP) membranes consisting of either single-walled carbon nanotubes (SWNTs) or multi-walled carbon nanotubes (MWNTs), and the antibiotic ciprofloxacin (cipro), is reported. The electrical, mechanical and morphological properties of these membranes have been characterised and are compared to those of the corresponding buckypaper membranes containing the surfactant Triton X-100 (Trix). Analysis of scanning electron microscopic images of the surfaces of SWNT/cipro and SWNT/Trix (Trix  =  Triton X-100) buckypapers revealed that the diameter of their surface pores was significantly smaller than that of the corresponding materials prepared using MWNTs. Similarly, the average internal pore diameter of both SWNT buckypapers was found to be smaller than that of their MWNT counterparts, after analysis of binding isotherms derived from nitrogen adsorption/desorption measurements performed on the materials. All four buckypaper membranes examined were found to be >99% effective for removing Escherichia coli (E. coli) from aqueous suspensions. However, buckypapers containing ciprofloxacin outperformed their counterparts containing the surfactant. Both MWNT buckypapers were more effective at preventing passage of E. coli than their analogues containing SWNTs, while fluorescence microscopic examination of stained membrane surfaces demonstrated that buckypapers composed of SWNTs had greater bactericidal properties

Evaluation of RTS,S/AS02A and RTS,S/AS01B in Adults in a High Malaria Transmission Area

This study advances the clinical development of the RTS,S/AS01B candidate malaria vaccine to malaria endemic populations. As a primary objective it compares the safety and reactogenicity of RTS,S/AS01B to the more extensively evaluated RTS,S/AS02A vaccine.A Phase IIb, single centre, double-blind, controlled trial of 6 months duration with a subsequent 6 month single-blind follow-up conducted in Kisumu West District, Kenya between August 2005 and August 2006. 255 healthy adults aged 18 to 35 years were randomized (1ratio1ratio1) to receive 3 doses of RTS,S/AS02A, RTS,S/AS01B or rabies vaccine (Rabipur; Chiron Behring GmbH) at months 0, 1, 2. The primary objective was the occurrence of severe (grade 3) solicited or unsolicited general (i.e. systemic) adverse events (AEs) during 7 days follow up after each vaccination.Both candidate vaccines had a good safety profile and were well tolerated. One grade 3 systemic AE occurred within 7 days of vaccination (RTS,S/AS01B group). No unsolicited AEs or SAEs were related to vaccine. A marked increase in anti-CS antibody GMTs was observed post Dose 2 of both RTS,S/AS01B (31.6 EU/mL [95% CI: 23.9 to 41.6]) and RTS,S/AS02A (16.7 EU/mL [95% CI: 12.9 to 21.7]). A further increase was observed post Dose 3 in both the RTS,S/AS01B (41.4 EU/mL [95% CI: 31.7 to 54.2]) and RTS,S/AS02A (21.4 EU/mL [95% CI: 16.0 to 28.7]) groups. Anti-CS antibody GMTs were significantly greater with RTS,S/AS01B compared to RTS,S/AS02A at all time points post Dose 2 and Dose 3. Both candidate vaccines produced strong anti-HBs responses. Vaccine efficacy in the RTS,S/AS01B group was 29.5% (95% CI: -15.4 to 56.9, p = 0.164) and in the RTS,S/AS02A group 31.7% (95% CI: -11.6 to 58.2, p = 0.128).Both candidate malaria vaccines were well tolerated over a 12 month surveillance period. A more favorable immunogenicity profile was observed with RTS,S/AS01B than with RTS,S/AS02A.Clinicaltrials.gov NCT00197054

Rationale and design of the Early valve replacement in severe ASYmptomatic Aortic Stenosis Trial

Background: Aortic valve replacement in asymptomatic severe aortic stenosis is controversial. The Early valve replacement in severe ASYmptomatic Aortic Stenosis (EASY-AS) trial aims to determine whether early aortic valve replacement improves clinical outcomes, quality of life and cost-effectiveness compared to a guideline recommended strategy of ‘watchful waiting’. Methods: In a pragmatic international, open parallel group randomized controlled trial (NCT04204915), 2844 patients with severe aortic stenosis will be randomized 1:1 to either a strategy of early (surgical or transcatheter) aortic valve replacement or aortic valve replacement only if symptoms or impaired left ventricular function develop, or other cardiac surgery becomes nessessary. Exclusion criteria include other severe valvular disease, planned cardiac surgery, ejection fraction &lt;50%, previous aortic valve replacement or life expectancy &lt;2 years. The primary outcome is a composite of cardiovascular mortality or heart failure hospitalization. The primary analysis will be undertaken when 663 primary events have accrued, providing 90% power to detect a reduction in the primary endpoint from 27.7% to 21.6% (hazard ratio 0.75). Secondary endpoints include disability-free survival, days alive and out of hospital, major adverse cardiovascular events and quality of life. Results: Recruitment commenced in March 2020 and is open in the UK, Australia, New Zealand, and Serbia. Feasibility requirements were met in July 2022, and the main phase opened in October 2022, with additional international centers in set-up. Conclusions: The EASY-AS trial will establish whether a strategy of early aortic valve replacement in asymptomatic patients with severe aortic stenosis reduces cardiovascular mortality or heart failure hospitalization and improves other important outcomes.</p