Bedaquiline-Pretomanid-Linezolid Regimens for Drug-Resistant Tuberculosis.

BACKGROUND: The bedaquiline-pretomanid-linezolid regimen has been reported to have 90% efficacy against highly drug-resistant tuberculosis, but the incidence of adverse events with 1200 mg of linezolid daily has been high. The appropriate dose of linezolid and duration of treatment with this agent to minimize toxic effects while maintaining efficacy against highly drug-resistant tuberculosis are unclear. METHODS: We enrolled participants with extensively drug-resistant (XDR) tuberculosis (i.e., resistant to rifampin, a fluoroquinolone, and an aminoglycoside), pre-XDR tuberculosis (i.e., resistant to rifampin and to either a fluoroquinolone or an aminoglycoside), or rifampin-resistant tuberculosis that was not responsive to treatment or for which a second-line regimen had been discontinued because of side effects. We randomly assigned the participants to receive bedaquiline for 26 weeks (200 mg daily for 8 weeks, then 100 mg daily for 18 weeks), pretomanid (200 mg daily for 26 weeks), and daily linezolid at a dose of 1200 mg for 26 weeks or 9 weeks or 600 mg for 26 weeks or 9 weeks. The primary end point in the modified intention-to-treat population was the incidence of an unfavorable outcome, defined as treatment failure or disease relapse (clinical or bacteriologic) at 26 weeks after completion of treatment. Safety was also evaluated. RESULTS: A total of 181 participants were enrolled, 88% of whom had XDR or pre-XDR tuberculosis. Among participants who received bedaquiline-pretomanid-linezolid with linezolid at a dose of 1200 mg for 26 weeks or 9 weeks or 600 mg for 26 weeks or 9 weeks, 93%, 89%, 91%, and 84%, respectively, had a favorable outcome; peripheral neuropathy occurred in 38%, 24%, 24%, and 13%, respectively; myelosuppression occurred in 22%, 15%, 2%, and 7%, respectively; and the linezolid dose was modified (i.e., interrupted, reduced, or discontinued) in 51%, 30%, 13%, and 13%, respectively. Optic neuropathy developed in 4 participants (9%) who had received linezolid at a dose of 1200 mg for 26 weeks; all the cases resolved. Six of the seven unfavorable microbiologic outcomes through 78 weeks of follow-up occurred in participants assigned to the 9-week linezolid groups. CONCLUSIONS: A total of 84 to 93% of the participants across all four bedaquiline-pretomanid-linezolid treatment groups had a favorable outcome. The overall risk-benefit ratio favored the group that received the three-drug regimen with linezolid at a dose of 600 mg for 26 weeks, with a lower incidence of adverse events reported and fewer linezolid dose modifications. (Funded by the TB Alliance and others; ZeNix ClinicalTrials.gov number, NCT03086486.).

Pathways to, and use of, sexual healthcare among Black Caribbean sexual health clinic attendees in England: evidence from cross-sectional bio-behavioural surveys.

BACKGROUND: In England, people of Black Caribbean (BC) ethnicity are disproportionately affected by sexually transmitted infections (STI). We examined whether differences in sexual healthcare behaviours contribute to these inequalities. METHODS: We purposively selected 16 sexual health clinics across England with high proportions of attendees of BC ethnicity. During May-September 2016, attendees at these clinics (of all ethnicities) completed an online survey that collected data on health service use and sexual behaviour. We individually linked these data to routinely-collected surveillance data. We then used multivariable logistic regression to compare reported behaviours among BC and White British/Irish (WBI) attendees (n = 627, n = 1411 respectively) separately for women and men, and to make comparisons by gender within these ethnic groups. RESULTS: BC women's sexual health clinic attendances were more commonly related to recent bacterial STI diagnoses, compared to WBI women's attendances (adjusted odds ratio, AOR 3.54, 95% CI 1.45-8.64, p = 0.009; no gender difference among BC attendees), while BC men were more likely than WBI men (and BC women) to report attending because of a partner's symptoms or diagnosis (AOR 1.82, 95% CI 1.14-2.90; AOR BC men compared with BC women: 4.36, 95% CI 1.42-13.34, p = 0.014). Among symptomatic attendees, BC women were less likely than WBI women to report care-seeking elsewhere before attending the sexual health clinic (AOR 0.60, 95% CI 0.38-0.97, p = 0.039). No ethnic differences, or gender differences among BC attendees, were observed in symptom duration, or reporting sex whilst symptomatic. Among those reporting previous diagnoses with or treatment for bacterial STI, no differences were observed in partner notification. CONCLUSIONS: Differences in STI diagnosis rates observed between BC and WBI ethnic groups were not explained by the few ethnic differences which we identified in sexual healthcare-seeking and use. As changes take place in service delivery, prompt clinic access must be maintained - and indeed facilitated - for those at greatest risk of STI, regardless of ethnicity.

Reduced telomere length is associated with fibrotic joint disease suggesting that impaired telomere repair contributes to joint fibrosis.

OBJECTIVE: Joint fibrosis affects many synovial joints (including hip, knee and shoulder) causing stiffness and pain. The mechanism of joint fibrosis remains unknown, although genetic factors may contribute. Defects in maintenance of telomere length resulting from impaired telomere repair have been shown to cause lung and liver fibrotic disease. Here we tested the hypothesis that joint fibrosis and other soft tissue fibrotic conditions are also associated with telomere length. PATIENTS AND METHODS: 5,200 participants in the TwinsUK registry had data on telomere length (measured by qPCR) and the traits of interest (hip and knee stiffness, total joint replacement (TJR, hip or knee) and fibrotic conditions (Dupuytren's disease, frozen shoulder). RESULTS: Multivariable logistic regression analyses showed a significant association between telomere length and fibrotic conditions (hip stiffness, knee stiffness and frozen shoulder, p = ≤0.002) even after taking age into account. No association was found between TJR and telomere length. CONCLUSION: These findings suggest that defects in telomere repair contribute to joint fibrosis, and that fibrosis shares a common mechanistic pathway in different organs. Therapeutic strategies to combat telomere shortening may offer novel treatments for fibrotic joint disease.

The Association Between Low Back Pain and Composition of IgG Glycome.

Low back pain (LBP) is a common debilitating condition which aetiology and pathogenesis are poorly understood. We carried out a first so far analysis of associations between LBP and plasma IgG N-glycome in a sample of 4511 twins from TwinsUK database assessed for LBP, lumbar disc degeneration (LDD) as its possible cause, and IgG-glycan levels. Using weighted correlation network analysis, we established a correlation between LBP and glycan modules featured by glycans that either promote or block antibody-dependent cell-mediated cytotoxicity (ADCC). The levels of four glycan traits representing two of those modules were statistically significantly different in monozygotic twins discordant for LBP. Also, the trend to higher prevalence of systemic inflammatory disorders was shown for twins with low level of fucosylated glycans and high level of non-fucosylated glycans. Core fucosylation of IgG is a "safety switch" reducing ADCC, thus our results suggest the involvement of ADCC and associated inflammation in pathogenesis of LBP. No correlation between LDD scores and glycans was found assuming that the inflammation may not be a part of LDD. These data provide a new insight into understanding the complex pathophysiology of LBP and suggest glycan levels as a possible biomarker for inflammation-related subtypes of LBP.

Joint Stiffness Is Heritable and Associated with Fibrotic Conditions and Joint Replacement.

OBJECTIVE: Joint stiffness is a common, debilitating, age-related symptom, which may be seen after total joint replacement (TJR). Stiffness also occurs in fibrotic conditions such as shoulder capsulitis and Dupuytren's contracture. We speculated that the two traits (TJR and fibrotic disease) are linked pathogenically. METHODS: Using the TwinsUK NIHR BRC BioResource we tested the hypotheses that 1) joint (hip and knee) stiffness, TJR (hip and knee), and fibrotic conditions are associated and 2) genetic factors contribute to them. RESULTS: Participating twins (n = 9718) had completed self-reported questionnaires on the traits of interest. All three traits were significantly associated with increasing age and body mass index (BMI), as well as female sex, on univariate analysis. Multivariable logistic regression analyses showed a significant association between TJR and joint stiffness (OR = 3.96, 95% confidence interval, CI 2.77-5.68) and between fibrotic conditions and joint stiffness (OR = 2.39, 1.74-3.29), adjusting for age, sex, BMI and twin relatedness. Monozygotic versus dizygotic intraclass correlations gave heritability estimates for TJR = 46% and joint stiffness = 32%. CONCLUSION: That fibrotic conditions, joint stiffness and TJR are significantly associated suggests a common disease process, possibly fibrosis, which is genetically mediated.

Conformational plasticity at the IgE-binding site of the B-cell receptor CD23.

IgE antibodies play a central role in allergic disease. They recognize allergens via their Fab regions, whilst their effector functions are controlled through interactions of the Fc region with two principal cell surface receptors, FcɛRI and CD23. Crosslinking of FcɛRI-bound IgE on mast cells and basophils by allergen initiates an immediate inflammatory response, while the interaction of IgE with CD23 on B-cells regulates IgE production. We have determined the structures of the C-type lectin "head" domain of CD23 from seven crystal forms. The thirty-five independent structures reveal extensive conformational plasticity in two loops that are critical for IgE binding.

Crystal structure of IgE bound to its B-cell receptor CD23 reveals a mechanism of reciprocal allosteric inhibition with high affinity receptor FcεRI.

The role of IgE in allergic disease mechanisms is performed principally through its interactions with two receptors, FcεRI on mast cells and basophils, and CD23 (FcεRII) on B cells. The former mediates allergic hypersensitivity, the latter regulates IgE levels, and both receptors, also expressed on antigen-presenting cells, contribute to allergen uptake and presentation to the immune system. We have solved the crystal structure of the soluble lectin-like "head" domain of CD23 (derCD23) bound to a subfragment of IgE-Fc consisting of the dimer of Cε3 and Cε4 domains (Fcε3-4). One CD23 head binds to each heavy chain at the interface between the two domains, explaining the known 2:1 stoichiometry and suggesting mechanisms for cross-linking membrane-bound trimeric CD23 by IgE, or membrane IgE by soluble trimeric forms of CD23, both of which may contribute to the regulation of IgE synthesis by B cells. The two symmetrically located binding sites are distant from the single FcεRI binding site, which lies at the opposite ends of the Cε3 domains. Structural comparisons with both free IgE-Fc and its FcεRI complex reveal not only that the conformational changes in IgE-Fc required for CD23 binding are incompatible with FcεRI binding, but also that the converse is true. The two binding sites are allosterically linked. We demonstrate experimentally the reciprocal inhibition of CD23 and FcεRI binding in solution and suggest that the mutual exclusion of receptor binding allows IgE to function independently through its two receptors.

The crystal structure of an avian IgY-Fc fragment reveals conservation with both mammalian IgG and IgE.

Avian IgY is closely related to an ancestor of both mammalian IgG and IgE and thus provides insights into the evolution of antibody structure and function. A recombinant fragment of IgY-Fc consisting of a dimer of the Cupsilon3 and Cupsilon4 domains, Fcupsilon3-4, was expressed and crystallized and its X-ray structure determined to 1.75 A resolution. Fcupsilon3-4 is the only nonmammalian Fc fragment structure determined to date and provides the first structural evidence for an ancient origin of antibody architecture. The Fcupsilon3-4 structure reveals features common to both IgE-Fc and IgG-Fc, and the implications for IgY binding to its receptor are discussed.

Adaptive protein evolution grants organismal fitness by improving catalysis and flexibility.

Protein evolution is crucial for organismal adaptation and fitness. This process takes place by shaping a given 3-dimensional fold for its particular biochemical function within the metabolic requirements and constraints of the environment. The complex interplay between sequence, structure, functionality, and stability that gives rise to a particular phenotype has limited the identification of traits acquired through evolution. This is further complicated by the fact that mutations are pleiotropic, and interactions between mutations are not always understood. Antibiotic resistance mediated by beta-lactamases represents an evolutionary paradigm in which organismal fitness depends on the catalytic efficiency of a single enzyme. Based on this, we have dissected the structural and mechanistic features acquired by an optimized metallo-beta-lactamase (MbetaL) obtained by directed evolution. We show that antibiotic resistance mediated by this enzyme is driven by 2 mutations with sign epistasis. One mutation stabilizes a catalytically relevant intermediate by fine tuning the position of 1 metal ion; whereas the other acts by augmenting the protein flexibility. We found that enzyme evolution (and the associated antibiotic resistance) occurred at the expense of the protein stability, revealing that MbetaLs have not exhausted their stability threshold. Our results demonstrate that flexibility is an essential trait that can be acquired during evolution on stable protein scaffolds. Directed evolution aided by a thorough characterization of the selected proteins can be successfully used to predict future evolutionary events and design inhibitors with an evolutionary perspective.

Soluble CD23 monomers inhibit and oligomers stimulate IGE synthesis in human B cells.

The low affinity IgE receptor, CD23, is implicated in IgE regulation and the pathogenesis of allergic disease. CD23 is a type II integral membrane protein, comprising a lectin "head," N-terminal "stalk," and C-terminal "tail" in the extracellular sequence. Endogenous proteases cleave CD23 in the stalk and the tail to release soluble fragments that either stimulate or inhibit IgE synthesis in human B cells. The molecular basis of these paradoxical activities is not understood. We have characterized three fragments of CD23, monomeric derCD23, monomeric exCD23, and oligomeric lzCD23. We show that the monomers inhibit and the oligomer stimulates IgE synthesis in human B cells after heavy chain switching to IgE. CD23 fragments could be targets for therapeutic intervention in allergic disease.

Asp-120 locates Zn2 for optimal metallo-beta-lactamase activity.

Metallo-beta-lactamases are zinc-dependent hydrolases that inactivate beta-lactam antibiotics, rendering bacteria resistant to them. Asp-120 is fully conserved in all metallo-beta-lactamases and is central to catalysis. Several roles have been proposed for Asp-120, but so far there is no agreed consensus. We generated four site-specifically substituted variants of the enzyme BcII from Bacillus cereus as follows: D120N, D120E, D120Q, and D120S. Replacement of Asp-120 by other residues with very different metal ligating capabilities severely impairs the lactamase activity without abolishing metal binding to the mutated site. A kinetic study of these mutants indicates that Asp-120 is not the proton donor, nor does it play an essential role in nucleophilic activation. Spectroscopic and crystallographic analysis of D120S BcII, the least active mutant bearing the weakest metal ligand in the series, reveals that this enzyme is able to accommodate a dinuclear center and that perturbations in the active site are limited to the Zn2 site. It is proposed that the role of Asp-120 is to act as a strong Zn2 ligand, locating this ion optimally for substrate binding, stabilization of the development of a partial negative charge in the beta-lactam nitrogen, and protonation of this atom by a zinc-bound water molecule.

Crystal structure of a human autoimmune complex between IgM rheumatoid factor RF61 and IgG1 Fc reveals a novel epitope and evidence for affinity maturation.

Rheumatoid factors (RF) are autoantibodies that recognize epitopes in the Fc region of immunoglobulin (Ig) G and that correlate with the clinical severity of rheumatoid arthritis (RA). Here we report the X-ray crystallographic structure, at 3 A resolution, of a complex between the Fc region of human IgG1 and the Fab fragment of a monoclonal IgM RF (RF61), derived from an RA patient and with a relatively high affinity for IgG Fc. In the complex, two Fab fragments bind to each Fc at epitopes close to the C terminus, and each epitope comprises residues from both Cgamma3 domains. A central role in the unusually hydrophilic epitope is played by the side-chain of Arg355, accounting for the subclass specificity of RF61, which recognizes IgG1,-2, and -3 in preference to IgG4, in which the corresponding residue is Gln355. Compared with a previously determined complex of a lower affinity RF (RF-AN) bound to IgG4 Fc, in which only residues at the very edge of the antibody combining site were involved in binding, the epitope bound by RF61 is centered in classic fashion on the axis of the V(H):V(L) beta-barrel. The complementarity determining region-H3 loop plays a key role, forming a pocket in which Arg355 is bound by two salt-bridges. The antibody contacts also involve two somatically mutated V(H) residues, reinforcing the suggestion of a process of antigen-driven maturation and selection for IgG Fc during the generation of this RF autoantibody.

Effect of pH on the active site of an Arg121Cys mutant of the metallo-beta-lactamase from Bacillus cereus: implications for the enzyme mechanism.

The zinc-dependent metallo-beta-lactamases are a group of bacterial enzymes that pose a threat to the future efficacy of present-day antibiotics. Their mechanism is poorly understood, and there are no clinically useful inhibitors. While most members of the group contain two tightly bound zinc ions in their active sites, the Bacillus cereus enzyme has a much lower affinity for its second zinc (Zn2), thought to be due to the presence of Arg121 immediately beneath the floor of the active site (cf. Cys/Ser/His121 in the bizinc enzymes). Crystal structures of the Arg121Cys mutant of the B. cereus 569/H/9 enzyme were solved at pH 7.0, 5.0, and 4.5, each in the presence of either 20 microM or 20 mM Zn(2+) to generate the mono- and bizinc forms, respectively. Surprisingly, the structure of the active site was unaffected by the mutation; a network of ordered water molecules replaced the interactions made by the arginine side chain, and the occupancy of Zn2 appeared minimally changed. As the pH was lowered, Zn2 moved away from one of its ligands, Asp120, but was "tracked" by two others, Cys221 and His263. Furthermore, the hydroxide ion (and proposed nucleophile for beta-lactam hydrolysis) was bound to Zn1 at pH 5 and above but absent at pH 4.5. This provides experimental evidence for an earlier proposed mechanism in which protonation of Asp120 and the Zn1-bound hydroxide are the two events that lead to the loss of activity at low pH.

The crystal structure of IgE Fc reveals an asymmetrically bent conformation.

The distinguishing structural feature of immunoglobulin E (IgE), the antibody responsible for allergic hypersensitivity, is the C epsilon 2 domain pair that replaces the hinge region of IgG. The crystal structure of the IgE Fc (constant fragment) at a 2.6-A resolution has revealed these domains. They display a distinctive, disulfide-linked Ig domain interface and are folded back asymmetrically onto the C epsilon 3 and C epsilon 4 domains, which causes an acute bend in the IgE molecule. The structure implies that a substantial conformational change involving C epsilon 2 must accompany binding to the mast cell receptor Fc epsilon RI. This may be the basis of the exceptionally slow dissociation rate of the IgE-Fc epsilon RI complex and, thus, of the ability of IgE to cause persistent allergic sensitization of mast cells.