Preclinical development of a bispecific TNFα/IL-23 neutralising domain antibody as a novel oral treatment for inflammatory bowel disease.

Anti-TNFα and anti-IL-23 antibodies are highly effective therapies for Crohn's disease or ulcerative colitis in a proportion of patients. V56B2 is a novel bispecific domain antibody in which a llama-derived IL-23p19-specific domain antibody, humanised and engineered for intestinal protease resistance, V900, was combined with a previously-described TNFα-specific domain antibody, V565. V56B2 contains a central protease-labile linker to create a single molecule for oral administration. Incubation of V56B2 with trypsin or human faecal supernatant resulted in a complete separation of the V565 and V900 monomers without loss of neutralising potency. Following oral administration of V900 and V565 in mice, high levels of each domain antibody were detected in the faeces, demonstrating stability in the intestinal milieu. In ex vivo cultures of colonic biopsies from IBD patients, treatment with V565 or V900 inhibited tissue phosphoprotein levels and with a combination of the two, inhibition was even greater. These results support further development of V56B2 as an oral therapy for IBD with improved safety and efficacy in a greater proportion of patients as well as greater convenience for patients compared with traditional monoclonal antibody therapies.

Ophthalmologic characteristics and outcomes of children with cortical visual impairment and cerebral palsy.

BACKGROUND: Cortical visual impairment (CVI) is the most common cause of pediatric visual impairment in developed countries, and cerebral palsy (CP) is diagnosed in approximately half of children with CVI. It is unknown whether children with CVI who also have CP (CVI+CP) have different characteristics and outcomes (with regard to visual acuity, strabismus, and response to strabismus surgery) than children with CVI without CP (CVI-CP). METHODS: The medical records of all children with CVI, with and without CP, evaluated at our institution between 2013 and 2019 were retrospectively reviewed. Presentation and outcomes of children with CVI+CP were compared to those with CVI-CP. RESULTS: A total of 151 children with CVI+CP and 153 children with CVI-CP were included. Children with CVI+CP were more likely to be diagnosed with significant refractive error (53.6% vs 41.2%; P = 0.03), optic atrophy (46.4% vs 32.7%; P = 0.01), and strabismus (82.8% vs 72.5%; P = 0.03) at presentation. Good ocular alignment after strabismus surgery was achieved in 30% of children with CVI+CP and 63.6% of children with CVI-CP (P = 0.48). Of 9 children with long-term (≥8 years) postoperative follow-up, 100% of CVI-CP patients achieved good outcomes compared with 0% of CVI+CP patients (P = 0.0079). Visual acuity at presentation and the percentage of patients who experienced improvement in visual acuity did not differ between groups. CONCLUSIONS: In our study cohort, children with CVI+CP had a higher likelihood of ophthalmic comorbidities and may have worse long-term strabismus surgery outcomes than children with CVI-CP.

Oral Anti-Tumour Necrosis Factor Domain Antibody V565 Provides High Intestinal Concentrations, and Reduces Markers of Inflammation in Ulcerative Colitis Patients.

V565 is an engineered TNFα-neutralising single domain antibody formulated into enteric coated mini-tablets to enable release in the intestine after oral administration as a possible oral treatment for inflammatory bowel disease (IBD). Following oral administration, ileal recovery of V565 was investigated in four patients with terminal ileostomy. Intestinal and systemic pharmacokinetics were measured in six patients with Crohn's disease and evidence of target engagement assessed in five patients with ulcerative colitis. Following oral administration, V565 was detected at micromolar concentrations in ileal fluid from the ileostomy patients and in stools of the Crohn's patients. In four of the five ulcerative colitis patients, biopsies taken after 7d dosing demonstrated V565 in the lamina propria with co-immunostaining on CD3+ T-lymphocytes and CD14+ macrophages. Phosphorylation of signalling proteins in biopsies taken after 7d oral dosing was decreased by approximately 50%. In conclusion, enteric coating of V565 mini-tablets provided protection in the stomach with gradual release in intestinal regions affected by IBD. Immunostaining revealed V565 tissue penetration and association with inflammatory cells, while decreased phosphoproteins after 7d oral dosing was consistent with V565-TNFα engagement and neutralising activity. Overall these results are encouraging for the clinical utility of V565 in the treatment of IBD.

Oral delivery of the anti-tumor necrosis factor α domain antibody, V565, results in high intestinal and fecal concentrations with minimal systemic exposure in cynomolgus monkeys.

OBJECTIVE: V565 is a novel oral anti-tumor necrosis factor (TNF)-α domain antibody being developed for topical treatment of inflammatory bowel disease (IBD) patients. Protein engineering rendered the molecule resistant to intestinal proteases. Here we investigate the formulation of V565 required to provide gastro-protection and enable optimal delivery to the lower intestinal tract in monkeys. METHODS: Enteric-coated V565 mini-tablets were prepared and dissolution characteristics tested in vitro. Oral dosing of monkeys with enteric-coated mini-tablets containing V565 and methylene blue dye enabled in vivo localization of mini-tablet dissolution. V565 distribution in luminal contents and feces was measured by enzyme-linked immunosorbent assay (ELISA). To mimic transit across the damaged intestinal epithelium seen in IBD patients an intravenous (i.v.) bolus of V565 was given to monkeys and pharmacokinetic parameters of V565 measured in serum and urine by ELISA. RESULTS: Enteric-coated mini-tablets resisted dissolution in 0.1 M HCl, before dissolving in a sustained release fashion at neutral pH. In orally dosed monkeys methylene blue intestinal staining indicated the jejunum and ileum as sites for mini-tablet dissolution. Measurements of V565 in monkey feces confirmed V565 survival through the intestinal tract. Systemic exposure after oral dosing was very low consistent with limited V565 mucosal penetration in healthy monkeys. The rapid clearance of V565 after i.v. dosing was consistent with renal excretion as the primary route for elimination of any V565 reaching the circulation. CONCLUSIONS: These results suggest that mini-tablets with a 24% Eudragit enteric coating are suitable for targeted release of orally delivered V565 in the intestine for topical treatment of IBD.

Preclinical Development of a Novel, Orally-Administered Anti-Tumour Necrosis Factor Domain Antibody for the Treatment of Inflammatory Bowel Disease.

TNFα is an important cytokine in inflammatory bowel disease. V565 is a novel anti-TNFα domain antibody developed for oral administration in IBD patients, derived from a llama domain antibody and engineered to enhance intestinal protease resistance. V565 activity was evaluated in TNFα-TNFα receptor-binding ELISAs as well as TNFα responsive cellular assays and demonstrated neutralisation of both soluble and membrane TNFα with potencies similar to those of adalimumab. Although sensitive to pepsin, V565 retained activity after lengthy incubations with trypsin, chymotrypsin, and pancreatin, as well as mouse small intestinal and human ileal and faecal supernatants. In orally dosed naïve and DSS colitis mice, high V565 concentrations were observed in intestinal contents and faeces and immunostaining revealed V565 localisation in mouse colon tissue. V565 was detected by ELISA in post-dose serum of colitis mice, but not naïve mice, demonstrating penetration of disrupted epithelium. In an ex vivo human IBD tissue culture model, V565 inhibition of tissue phosphoprotein levels and production of inflammatory cytokine biomarkers was similar to infliximab, demonstrating efficacy when present at the disease site. Taken together, results of these studies provide confidence that oral V565 dosing will be therapeutic in IBD patients where the mucosal epithelial barrier is compromised.

Aspergillus thromboembolism from a mycotic ascending aortic pseudoaneurysm.

This case report describes an immune-competent patient with acute upper extremity ischemia caused by thromboembolism from an Aspergillus-infected ascending aortic pseudoaneurysm. Efforts to identify the source of an acute arterial thromboembolic occlusion should be made, and a high index of suspicion for mycotic infection should be maintained in patients with an atypical presentation, such as fevers of unknown origin. Additional measures, such as pathologic examination of thromboembolic debris, blood cultures, and positron emission tomography, should be performed to identify the etiology in these unexplained situations.

The Clostridium difficile spo0A gene is a persistence and transmission factor.

Clostridium difficile is a major cause of chronic antibiotic-associated diarrhea and a significant health care-associated pathogen that forms highly resistant and infectious spores. Spo0A is a highly conserved transcriptional regulator that plays a key role in initiating sporulation in Bacillus and Clostridium species. Here, we use a murine model to study the role of the C. difficile spo0A gene during infection and transmission. We demonstrate that C. difficile spo0A mutant derivatives can cause intestinal disease but are unable to persist within and effectively transmit between mice. Thus, the C. difficile Spo0A protein plays a key role in persistent infection, including recurrence and host-to-host transmission in mice.

Plume detachment and recession times in fractured rock.

The influence of source zone concentration reduction on solute plume detachment and recession times in fractured rock was investigated using new semianalytical solutions to transient solute transport in the presence of advection, dispersion, sorption, matrix diffusion, and first-order decay. Novel aspects of these solutions are: (1) the source zone concentration behavior is simulated using a constant concentration with the option for either an instantaneous reduction to zero concentration or an exponentially decaying source zone concentration initiated at some time (t*) after the source is introduced, and (2) different biodegradation rates in the fracture and rock matrix. These solutions were applied for sandstone bedrock and revealed that biodegradation in the matrix, not the fracture, may be the most significant attenuation mechanism and therefore may dictate remediation time scales. Also, instantaneous and complete source concentration reduction in aged plumes may not be beneficial with respect to plume response because back-diffusion can sustain plume migration for long periods of time. Moderate source zone concentration reduction has a similar impact on the rate of advance of the leading edge of the plume as aggressive concentration reduction. If the source zone concentration reduction half-life is less than the plume decay half-life, then volatile organic compound (VOC) mass sequestered in the rock matrix will ultimately dictate plume persistence and not the presence of the source zone.

Concentration rebound following in situ chemical oxidation in fractured clay.

A two-dimensional, transient-flow, and transport numerical model was developed to simulate in situ chemical oxidation (ISCO) of trichloroethylene and tetrachloroethylene by potassium permanganate in fractured clay. This computer model incorporates dense, nonaqueous phase liquid dissolution, reactive aquifer material, multispecies matrix diffusion, and kinetic formulations for the oxidation reactions. A sensitivity analysis for two types of parameters, hydrogeological and engineering, including matrix porosity, matrix organic carbon, fracture aperture, potassium permanganate dosage, and hydraulic gradient, was conducted. Remediation metrics investigated were the relative rebound concentrations arising from back diffusion and percent mass destroyed. No well-defined correlation was found between the magnitude of rebound concentrations during postremedy monitoring and the amount of contaminant mass destroyed during the application. Results indicate that all investigated parameters affect ISCO remediation in some form. Results indicate that when advective transport through the fracture is dominant relative to diffusive transport into the clay matrix (large System Peclet Number), permanganate is more likely to be flushed out of the system and treatment is not optimal. If the System Peclet Number is too small, indicating that diffusion into the matrix is dominant relative to advection through the fracture, permanganate does not traverse the entire fracture, leading to postremediation concentration rebound. Optimal application of ISCO requires balancing advective transport through the fracture with diffusive transport into the clay matrix.

Pharmacological properties of the enhanced-affinity glucocorticoid fluticasone furoate in vitro and in an in vivo model of respiratory inflammatory disease.

Fluticasone furoate (FF) is a novel enhanced-affinity glucocorticoid that has been developed as topical therapy for allergic rhinitis. The pharmacological properties of FF have been investigated using a number of in vitro experimental systems. FF demonstrated very potent glucocorticoid activity in several key pathways downstream of the glucocorticoid receptor (GR) as follows: the transrepression nuclear factor-kappaB (NF-kappaB) pathway, the transactivation glucocorticoid response element pathway, and inhibition of the proinflammatory cytokine tumor necrosis factor-alpha. Furthermore, FF showed the greatest potency compared with other glucocorticoids for preserving epithelial integrity and reducing epithelial permeability in response to protease- and mechanical-induced cell damage. FF showed a 30- to >330,000-fold selectivity for GR-mediated inhibition of NF-kappaB vs. the other steroid hormone receptors, substantially better than a number of other clinically used glucocorticoids. In studies examining the respiratory tissue binding properties of glucocorticoids, FF had the largest cellular accumulation and slowest rate of efflux compared with other clinically used glucocorticoids, consistent with greater tissue retention. The in vivo anti-inflammatory activity of FF was assessed in the Brown Norway rat ovalbumin-induced lung eosinophilial model of allergic lung inflammation. At a dose of only 30 microg, FF achieved almost total inhibition of eosinophil influx in the lung, an inhibition that was greater than that seen with the same dose of fluticasone propionate. In conclusion, the potent and selective pharmacological profile of FF described here could deliver an effective, safe, and sustained topical treatment of respiratory inflammatory diseases such as allergic rhinitis and asthma.

On the use and error of approximation in the Domenico (1987) solution.

A mathematical solution for solute transport in a three-dimensional porous medium with a patch source under steady-state, uniform ground water flow conditions was developed by Domenico (1987). The solution derivation strategy used an approximate approach to solve the boundary value problem, resulting in a nonexact solution. Variations of the Domenico (1987) solution are incorporated into the software programs BIOSCREEN and BIOCHLOR, which are frequently used to evaluate subsurface contaminant transport problems. This article mathematically elucidates the error in the approximation and presents simulations that compare different versions of the Domenico (1987) solution to an exact analytical solution to demonstrate the potential error inherent in the approximate expressions. Results suggest that the accuracy of the approximate solutions is highly variable and dependent on the selection of input parameters. For solute transport in a medium-grained sand aquifer, the Domenico (1987) solution underpredicts solute concentrations along the centerline of the plume by as much as 80% depending on the case of interest. Increasing the dispersivity, time, or dimensionality of the system leads to increased error. Because more accurate exact analytical solutions exist, we suggest that the Domenico (1987) solution, and its predecessor and successor approximate solutions, need not be employed as the basis for screening tools at contaminated sites.

CXCL13 neutralization reduces the severity of collagen-induced arthritis.

OBJECTIVE: To investigate the role of CXCL13 in the development and pathogenesis of collagen-induced arthritis (CIA), and to determine the mechanisms involved in the modulation of arthritogenic response by CXCL13 neutralization. METHODS: Mice were immunized with type II collagen (CII) and treated with anti-CXCL13 or control antibodies during boosting. Mice were monitored for the development and severity of arthritis. The effects of CXCL13 neutralization on immune response to CII were evaluated by cytokine production by CII-specific T cells and CII-specific antibody production. Follicular response in the spleen and in synovial tissue was determined by in situ immunohistology. RESULTS: Mice receiving neutralizing antibodies to CXCL13 developed significantly less severe arthritis compared with mice injected with phosphate buffered saline or control antibodies. Follicular response both in the spleen and in synovial tissue was inhibited by anti-CXCL13 treatment. Injection with anti-CXCL13 antibodies did not significantly affect antigen-specific recall lymphocyte proliferation or type 1 cytokine production in vitro. Antibody response specific to CII was not inhibited by anti-CXCL13 treatment. However, anti-CXCL13 treatment induced significantly higher levels of interleukin-10 production after in vitro CII stimulation. CONCLUSION: Neutralization of CXCL13 inhibits the development of CIA and reduces follicular response in both lymphoid and nonlymphoid tissues. These findings may have important implications regarding the pathogenesis and treatment of autoimmune arthritis.

Decreased distribution of lung epithelial junction proteins after intratracheal antigen or lipopolysaccharide challenge: correlation with neutrophil influx and levels of BALF sE-cadherin.

Distribution of airway junctional complex proteins after antigen or lipopolysaccharide challenge in sensitized or naive mice, respectively, was investigated. E-cadherin immunoreactivity was detected continuously along neighboring epithelial cell borders and between adjacent alveolar epithelial cells in naive and saline-challenged mice. Occludin and ZO-1 immunoreactivity were observed in the tight junction areas. Both challenges induced changes in epithelial morphology and phenotype, accompanied initially by focal loss of epithelial E-cadherin that increased in size with time and number of allergen challenges. Allergen challenge also led to focal loss of occludin and ZO-1. Western blot analysis revealed increased levels of sE-cadherin in lavage fluid after either challenge, and this increase correlated with lavage neutrophil numbers (P = 0.002). Immunocytochemistry of lavage cells 6 h after either challenge revealed E-cadherin epitopes within cytoplasmic vacuoles of neutrophils, the major cell type. In contrast, peripheral blood neutrophils or tissue neutrophils before epithelial transmigration were negative, suggesting that in airway inflammation, E-cadherin extracellular domain is cleaved by neutrophils during epithelial penetration, instigating the destabilization of adherens and tight junctions. This junctional deterioration could lead to a progressive decrease in epithelial integrity and induce alterations in epithelial morphology, with consequent enhanced paracellular transit of antigens and pathogens.