Engineering the Fab fragment of the anti-IgE omalizumab to prevent Fab crystallization and permit IgE-Fc complex crystallization

Immunoglobulin E (IgE) plays a central role in the allergic response, in which cross-linking of allergen by Fc[epsilon]RI-bound IgE triggers mast cell and basophil degranulation and the release of inflammatory mediators. The high-affinity interaction between IgE and Fc[epsilon]RI is a long-standing target for therapeutic intervention in allergic disease. Omalizumab is a clinically approved anti-IgE monoclonal antibody that binds to free IgE, also with high affinity, preventing its interaction with Fc[epsilon]RI. All attempts to crystallize the pre-formed complex between the omalizumab Fab and the Fc region of IgE (IgE-Fc), to understand the structural basis for its mechanism of action, surprisingly failed. Instead, the Fab alone selectively crystallized in different crystal forms, but their structures revealed intermolecular Fab/Fab interactions that were clearly strong enough to disrupt the Fab/IgE-Fc complexes. Some of these interactions were common to other Fab crystal structures. Mutations were therefore designed to disrupt two recurring packing interactions observed in the omalizumab Fab crystal structures without interfering with the ability of the omalizumab Fab to recognize IgE-Fc; this led to the successful crystallization and subsequent structure determination of the Fab/IgE-Fc complex. The mutagenesis strategy adopted to achieve this result is applicable to other intractable Fab/antigen complexes or systems in which Fabs are used as crystallization chaperones

Allosteric mechanism of action of the therapeutic anti-IgE antibody omalizumab

Immunoglobulin E and its interactions with receptors FcϵRI and CD23 play a central role in allergic disease. Omalizumab, a clinically approved therapeutic antibody, inhibits the interaction between IgE and FcϵRI, preventing mast cell and basophil activation, and blocks IgE binding to CD23 on B cells and antigen-presenting cells. We solved the crystal structure of the complex between an omalizumab-derived Fab and IgE-Fc, with one Fab bound to each Cϵ3 domain. Free IgE-Fc adopts an acutely bent structure, but in the complex it is only partially bent, with large-scale conformational changes in the Cϵ3 domains that inhibit the interaction with FcϵRI. CD23 binding is inhibited sterically due to overlapping binding sites on each Cϵ3 domain. Studies of omalizumab Fab binding in solution demonstrate the allosteric basis for FcϵRI inhibition and, together with the structure, reveal how omalizumab may accelerate dissociation of receptor-bound IgE from FcϵRI, exploiting the intrinsic flexibility and allosteric potential of IgE

52-week efficacy and safety of telbivudine with conditional tenofovir intensification at week 24 in HBeAg-positive chronic Hepatitis B

Background and Aims: The Roadmap concept is a therapeutic framework in chronic hepatitis B for the intensification of nucleoside analogue monotherapy based on early virologic response. The efficacy and safety of this approach applied to telbivudine treatment has not been investigated. Methods: A multinational, phase IV, single-arm open-label study (ClinicalTrials.gov ID NCT00651209) was undertaken in HBeAg-positive, nucleoside-naive adult patients with chronic hepatitis B. Patients received telbivudine (600 mg once-daily) for 24 weeks, after which those with undetectable serum HBV DNA (<300 copies/mL) continued to receive telbivudine alone while those with detectable DNA received telbivudine plus tenofovir (300 mg once-daily). Outcomes were assessed at Week 52. Results: 105 patients commenced telbivudine monotherapy, of whom 100 were included in the efficacy analysis. Fifty-five (55%) had undetectable HBV DNA at Week 24 and continued telbivudine monotherapy; 45 (45%) received tenofovir intensification. At Week 52, the overall proportion of undetectable HBV DNA was 93% (93/100) by last-observation-carried-forward analysis (100% monotherapy group, 84% intensification group) and no virologic breakthroughs had occurred. ALT normalization occurred in 77% (87% monotherapy, 64% intensification), HBeAg clearance in 43% (65% monotherapy, 16% intensification), and HBeAg seroconversion in 39% (62% monotherapy, 11% intensification). Six patients had HBsAg clearance. Myalgia was more common in the monotherapy group (19% versus 7%). No decrease in the mean glomerular filtration rate occurred in either treatment group at Week 52. Conclusions: Telbivudine therapy with tenofovir intensification at Week 24, where indicated by the Roadmap strategy, appears effective and well tolerated for the treatment of chronic hepatitis B. Trial Registration: ClinicalTrials.gov NCT0065120

Campylobacter pylori is not associated with gastroparesis

There is a high incidence of Campylobacter pylori in the gastric mucosa of patients with duodenal ulcer, gastric ulcer, and nonulcer dyspepsia. Factors that lead to development of this infection are unknown. We hypothesized that delayed solid-phase gastric emptying, a condition characterized by antral stasis, might predispose to Campylobacter pylori infection. We prospectively studied 51 patients with symptoms of gastroparesis using a solid-phase gastric emptying study and upper endoscopy. Patients were excluded if they had predominant symptoms of epigastric pain or an abnormal endoscopy. Three biopsies were obtained from the antrum and stained with H&E. When any inflammation was present, a Warthin-Starry stain was also performed. These were blindly examined for chronic inflammation, activity, and presence of Campylobacter pylori. Campylobacter pylori was not more common in patients with gastroparesis, documented by delayed gastric emptying, than in patients with a normal emptying study. On the contrary, there was a significantly lower incidence of Campylobacter pylori in those with delayed emptying compared to those with normal emptying (5% vs 31% , P<0.05). Gastritis activity correlated closely with Campylobacter presence. Inactive chronic gastritis with Campylobacter was equally common in those with delayed or normal gastric emptying. Diabetics were no more likely to harbor Campylobacter pylori than nondiabetics (16% vs 25%). The 5% incidence of Campylobacter in the gastroparesis group is less than, but approaches, that previously reported in asymptomatic controls. The 31% incidence of Campylobacter in the group with symptoms of gastroparesis but normal gastric emptying approaches that reported for nonulcer dyspepsia. Our data suggest that gastroparesis does not predispose to Campylobacter pylori infection or histologic chronic gastritis .Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44408/1/10620_2005_Article_BF01540043.pd

Multi-level evidence of an allelic hierarchy of USH2A variants in hearing, auditory processing and speech/language outcomes.

Language development builds upon a complex network of interacting subservient systems. It therefore follows that variations in, and subclinical disruptions of, these systems may have secondary effects on emergent language. In this paper, we consider the relationship between genetic variants, hearing, auditory processing and language development. We employ whole genome sequencing in a discovery family to target association and gene x environment interaction analyses in two large population cohorts; the Avon Longitudinal Study of Parents and Children (ALSPAC) and UK10K. These investigations indicate that USH2A variants are associated with altered low-frequency sound perception which, in turn, increases the risk of developmental language disorder. We further show that Ush2a heterozygote mice have low-level hearing impairments, persistent higher-order acoustic processing deficits and altered vocalizations. These findings provide new insights into the complexity of genetic mechanisms serving language development and disorders and the relationships between developmental auditory and neural systems

Influence of the incremental step size in work rate on exercise response and gas exchange in patients with pulmonary hypertension

<p>Abstract</p> <p>Background</p> <p>Cardiopulmonary exercise testing (CPET) has become increasingly important as a routine procedure in daily clinical work. So far, it is generally accepted that an individualized exercise protocol with exercise duration of 6 to 12 minutes is preferable to assess maximal exercise performance. The aim of this study was to compare an individualized NYHA adapted exercise protocol with a fixed standard protocol in patients with severe pulmonary arterial hypertension.</p> <p>Methods</p> <p>Twenty-two patients (17 female, 5 male; mean age 49 ± 14 yrs) underwent symptom limited CPET on a bicycle. On two consecutive days each subject performed a stepwise CPET according to a modified Jones protocol (16 Watt per minute stages) as well as an individualized NYHA adapted protocol with 5 or 10 Watt/min stages in a randomized order. Oxygen uptake at peak exercise (peakVO₂) and anaerobic threshold (VO₂AT), maximal ventilation (VE), breathing reserve (VE/MVV), ventilatory efficiency (VE vs. VCO₂slope), exercise time, maximal power and work rate were assessed and compared between both protocols.</p> <p>Results</p> <p>Comparing both, adapted NYHA protocol and standardized Jones protocol, we found significant differences in maximal power (56.7 ± 19 W vs. 74 ± 18 W; p < 0.001) and exercise time (332 ± 107 sec. vs. 248 ± 72 sec.; p < 0.001). In contrast, no significant differences were obvious comparing both protocols concerning work rate, VE, VE/MVV, peakVO₂, VO₂AT and VE vs. VCO₂slope.</p> <p>Conclusion</p> <p>Variations of incremental step size during CPET significantly affect exercise time and maximal power, whereas relevant parameters for clinical judgement and prognosis such as oxygen uptake, ventilation and ventilatory efficiency remain unchanged. These findings have practical implications for the exercise evaluation of patients with pulmonary hypertension. To reach maximal results for ventilation, oxygen uptake and gas exchange an individualization of incremental step size appears not to be mandatory.</p