A temporal thymic selection switch and ligand binding kinetics constrain neonatal Foxp3+ Treg cell development.

The neonatal thymus generates Foxp3+ regulatory T (tTreg) cells that are critical in controlling immune homeostasis and preventing multiorgan autoimmunity. The role of antigen specificity on neonatal tTreg cell selection is unresolved. Here we identify 17 self-peptides recognized by neonatal tTreg cells, and reveal ligand specificity patterns that include self-antigens presented in an age- and inflammation-dependent manner. Fate-mapping studies of neonatal peptidyl arginine deiminase type IV (Padi4)-specific thymocytes reveal disparate fate choices. Neonatal thymocytes expressing T cell receptors that engage IAb-Padi4 with moderate dwell times within a conventional docking orientation are exported as tTreg cells. In contrast, Padi4-specific T cell receptors with short dwell times are expressed on CD4+ T cells, while long dwell times induce negative selection. Temporally, Padi4-specific thymocytes are subject to a developmental stage-specific change in negative selection, which precludes tTreg cell development. Thus, a temporal switch in negative selection and ligand binding kinetics constrains the neonatal tTreg selection window.

Effect of CDR3 sequences and distal V gene residues in regulating TCR-MHC contacts and ligand specificity.

The mature T cell repertoire has the ability to orchestrate immunity to a wide range of potential pathogen challenges. This ability stems from thymic development producing individual T cell clonotypes that express TCRs with unique patterns of Ag reactivity. The Ag specificity of TCRs is created from the combinatorial pairing of one of a set of germline encoded TCR Vα and Vß gene segments with randomly created CDR3 sequences. How the amalgamation of germline encoded and randomly created TCR sequences results in Ag receptors with unique patterns of ligand specificity is not fully understood. Using cellular, biophysical, and structural analyses, we show that CDR3α residues can modulate the geometry in which TCRs bind peptide-MHC (pMHC), governing whether and how germline encoded TCR Vα and Vß residues interact with MHC. In addition, a CDR1α residue that is positioned distal to the TCR-pMHC binding interface is shown to contribute to the peptide specificity of T cells. These findings demonstrate that the specificity of individual T cell clonotypes arises not only from TCR residues that create direct contacts with the pMHC, but also from a collection of indirect effects that modulate how TCR residues are used to bind pMHC.

Bonding in singlet and triplet butalene: insights from spin-coupled theory.

Spin-coupled (SC, equivalent to full generalized valence bond) calculations for the (1)A(g) ground state of butalene at its optimal D(2h) planar geometry show that cross-ring Dewar-like modes of spin coupling are of comparable importance to the more usually considered Kekulé-like modes. There are marked similarities to the SC description of one of the isomers of benzo[1,2:4,5]dicyclobutadiene. A complication for both of these systems is the existence of SC solutions in which some of the orbitals resemble in- and out-of-phase combinations of semilocalized atom-centered orbitals. The lowest triplet state, for which a nonplanar C(2v) geometry is preferred, is somewhat more straightforward to analyze: the SC description of the (3)B2 state is dominated by a very simple pattern of two π bonds and two well-localized triplet-coupled orbitals.

Spin-coupled theory for 'N electrons in M orbitals' active spaces.

Spin-coupled (SC) theory, an ab initio valence bond (VB) approach which uses a compact and an easy-to-interpret single-orbital product wave function comparable in quality to a 'N in N' complete-active-space self-consistent field [CASSCF(N,N)] construction, is extended to 'N in M' (N ≠ M) active spaces. The SC(N,M) wave function retains the essential features of the original SC model: It involves just the products of nonorthogonal orbitals covering all distributions of N electrons between M orbitals in which as few orbitals as possible, |N ­ M|, are doubly occupied (for N > M) or missing (for N < M) and all other orbitals are singly occupied; each of these products is combined with a flexible spin function which allows any mode of coupling of the spins of the orbitals within the product. The SC(N,M) wave function remains much more compact than a CASSCF(N,M) construction; for example, the SC(6,7) wave function includes 35 configuration state functions (CSFs) as opposed to the 490 CSFs in the CASSCF case. The essential features of the SC(N,M) method are illustrated through a SC(6,5) calculation on the cyclopentadienyl anion, C5H5(­), and a SC(6,7) calculation on the tropylium cation, C7H7(+). The SC(6,5) and SC(6,7) wave functions for C5H5(­) and C7H7(+) are shown to provide remarkably clear modern VB models for the electronic structures of these aromatic cyclic ions which closely resemble the well-known SC model of benzene and yet recover almost all of the correlation energy included in the corresponding CASSCF(6,5) and CASSCF(6,7) wave functions: over 97% in the case of C5H5(­) and over 95% in the case of C7H7(+).

Cas9-derived peptides presented by MHC Class II that elicit proliferation of CD4+ T-cells.

CRISPR-Cas9 mediated genome editing offers unprecedented opportunities for treating human diseases. There are several reports that demonstrate pre-existing immune responses to Cas9 which may have implications for clinical development of CRISPR-Cas9 mediated gene therapy. Here we use 209 overlapping peptides that span the entire sequence of Staphylococcus aureus Cas9 (SaCas9) and human peripheral blood mononuclear cells (PBMCs) from a cohort of donors with a distribution of Major Histocompatibility Complex (MHC) alleles comparable to that in the North American (NA) population to identify the immunodominant regions of the SaCas9 protein. We also use an MHC Associated Peptide Proteomics (MAPPs) assay to identify SaCas9 peptides presented by MHC Class II (MHC-II) proteins on dendritic cells. Using these two data sets we identify 22 SaCas9 peptides that are both presented by MHC-II proteins and stimulate CD4+ T-cells.

P-glycoprotein-like protein, a possible genetic marker for ivermectin resistance selection in Onchocerca volvulus.

Ivermectin (IVM) is the only safe drug for mass-treatment of onchocerciasis. IVM resistance has been reported in gastrointestinal nematode parasites of animals. A reduction in response to IVM in Onchocerca volvulus could have significant consequences for the onchocerciasis control programs. We have found evidence that, in O. volvulus, repeated IVM treatment selects for specific alleles, of P-glycoprotein-like protein (PLP), a half-sized ABC transporter. In this study, O. volvulus samples were derived from a clinical trial in Cameroon, in which patients were sampled before, and following 3 years (1994-1997) of IVM treatments. There were four treatment groups: 150 microg/kg (1 x p.a. or 4 x p.a.) and 800 microg/kg (1 x p.a. or 4 x p.a.). DNA of O. volvulus macrofilariae was genotyped over a 476bp region of the PLP gene and at two control genes. Of the six polymorphic positions found in the PLP amplicon, three of them showed significant selection after 4 x p.a. treatment with IVM (total of 13 IVM treatments) in female worms, and one of the same single nucleotide polymorphisms (SNPs) showed significant selection in the male worms. One of the selected SNPs in the female worms caused an amino acid coding change in the putative protein sequence. We found a clear selection of some genotypes, a high SNPs association and a loss of polymorphism following 4 x p.a. treatment with IVM. These PLP SNPs and genotypes could be useful markers to follow selection for IVM resistance in the field.

Role of Tyr(356(7.43)) and Ser(190(4.57)) in antagonist binding in the rat beta1-adrenergic receptor.

Site-directed mutagenesis and photoaffinity labeling experiments suggest the existence of at least two distinct binding orientations for aryloxypropanolamine competitive antagonists in the beta-adrenergic receptor (beta-AR), one where the aryloxy moiety is located near transmembrane alpha-helix 7 (tm 7) and another where it is near tm 5. To explore a hydrophobic pocket involving tms 1, 2, 3, and 7 for potential aryloxy interaction sites, we selected Tyr(356(7.43)) and Trp(134(3.28)) in the rat beta(1)-AR for site-directed mutagenesis studies. Ser(190(4.57)) was also investigated, as the equivalent residues are known antagonist interaction sites in the muscarinic M(1) and the dopamine D(2) receptors. Binding affinities (pK(i)) of a series of structurally diverse aryloxypropanolamine competitive antagonists were determined for wild type and Y356A, Y356F, W134A, and S190A mutant rat beta(1)-ARs stably expressed in Chinese hamster ovary cells. To visualize possible antagonist/receptor interactions, the compounds were docked into a three-dimensional model of the wild-type rat beta(1)-AR. The results indicate that Tyr(356(7.43)) is an important aromatic interaction site for five of the eight competitive antagonists studied, whereas none of the compounds appeared to interact directly with Trp(134(3.28)). Only two of the competitive antagonists interacted with Ser(190(4.57)) on tm 4. Overall, the results extend our understanding of how beta(1)-AR competitive antagonists bind to the hydrophobic pocket involving tms 1, 2, 3, and 7; highlight the importance of Tyr(356(7.43)) in this binding pocket; and demonstrate the involvement of tm 4 in competitive antagonist binding.

A randomized, double-blind, controlled trial of the effects of ivermectin at normal and high doses, given annually or three-monthly, against Onchocerca volvulus: ophthalmological results.

A three-year randomized, controlled, double-blind trial was conducted in Cameroon to determine whether ivermectin, given at three-monthly intervals and/or at high doses (800 microg/kg), had a greater effect on adult Onchocerca volvulus than standard doses (150 microg/kg annually). As several patients complained of transitory subjective visual problems after treatment, some of them being of an unexpected type, we organized two series of detailed ophthalmological examinations to evaluate whether they were associated with ocular lesions. Analysis showed that these complaints were significantly more frequent in the two groups treated with high doses of ivermectin than in the reference group. In the ophthalmological examinations, the only differences recorded between the groups were a lower prevalence and mean number of microfilariae in the anterior chamber in the groups treated three-monthly, and, at the first examination round, a higher prevalence of early lesions of the iris in the group treated at high doses annually. These findings do not allow us to explain the cause of the transitory ocular complaints, nor why they were more frequent in the groups treated at high doses. However, one may conclude that using doses of ivermectin higher than the standard one should be considered with caution.

The effects of long-term community level treatment with ivermectin (Mectizan) on adult Onchocerca volvulus in Latin America.

The objective of this study was to examine nodules from Mexico, Guatemala, and Ecuador collected over a one-year period (2001) to determine the effects of semi-annual ivermectin treatments on Onchocerca volvulus macrofilarial populations. Nodules were sectioned, stained with hematoxylin and eosin, and histologic findings were compared between countries and with historical data prior to the introduction of ivermectin into the region. Nodules from Ecuador had 10 times more dead or moribund worms than the historical control (66.6% versus 6.5%); nodules from patients from Mexico and Guatemala did not differ from the control. More than 80% of the female worms in each country were uninseminated and producing unfertilized oocytes. Nodules containing males differed in each country from the historical control (P < 0.0001), with presence of males ranging from 19.7% in Mexico to 13.6% in Ecuador versus 73% in the control. Nodules with females producing active microfilariae ranged from 7.8% (Mexico) to 2.7% (Ecuador) versus 60% in the historical control (P < 0.0001). Nodules from Ecuador and Mexico were significantly smaller in size than those from Guatemala or historical controls (P < 0.0005). These results depict a deteriorating condition of adult O. volvulus populations in Mexico, Guatemala and Ecuador, indicating that semi-annual ivermectin treatment of >/=6 years has had a profound effect on survival and reproduction of this species.

Adverse systemic reactions to treatment of onchocerciasis with ivermectin at normal and high doses given annually or three-monthly.

In Cameroon, a 3-year randomized, double-blind controlled trial was conducted to determine if ivermectin, given at 3-monthly intervals and/or at high doses (800 microg/kg), had a greater effect on adult Onchocerca volvulus than standard annual doses of 150 microg/kg. Adverse reactions were recorded and analysed in a logistic regression model with random effects to assess the influence of the dose and rhythm of treatment on their occurrence. After the first dose, 3-monthly treatment was associated with a clearly reduced risk of reactions, especially oedematous swellings, pruritus and back-pain. Oedematous swellings and subjective ocular troubles were found to be associated with high doses of ivermectin. These results reinforce former parasitological conclusions that it would be desirable to evaluate the feasibility and effects on transmission of large-scale 3-monthly treatments with standard doses of ivermectin for onchocerciasis control. Owing to the unexpected ocular reactions, the use of high doses to counteract any future resistance of O. volvulus to ivermectin should be considered with caution.

Quantum chemical study of the intermediate complex required for iron-mediated reactivity and antimalarial activity of dispiro-1,2,4-trioxolanes.

Quantum chemical methods were used to obtain a structure for the peroxide-iron intermediate complex required for the inner-sphere reduction of dispiro-1,2,4-trioxolane antimalarials. Investigation of this biologically important interaction with iron(II) allows further understanding of the mechanisms of action and clearance of this promising new class of fully synthetic peroxide antimalarials. UHF, B3LYP and B3LYP//MP2 calculations were undertaken to provide structural and energetic information about the coordination complex of iron(II) with five representative trioxolanes, ranging in both iron-mediated reactivity and antimalarial activity. Significant energy differences were observed between the conformational isomers of these trioxolanes, indicating the importance of steric interactions between the iron complex ligands and the trioxolane substituents. These calculations may explain the slower iron-mediated reaction rates of trioxolanes that preferentially adopt a conformation that sterically shields the peroxide bond. The relationship between antimalarial activity and accessibility of the peroxide bond to iron has also been demonstrated for these trioxolanes.

Genetic selection of low fertile Onchocerca volvulus by ivermectin treatment.

BACKGROUND: Onchocerca volvulus is the causative agent of onchocerciasis, or "river blindness". Ivermectin has been used for mass treatment of onchocerciasis for up to 18 years, and recently there have been reports of poor parasitological responses to the drug. Should ivermectin resistance be developing, it would have a genetic basis. We monitored genetic changes in parasites obtained from the same patients before use of ivermectin and following different levels of ivermectin exposure. METHODS AND FINDINGS: O. volvulus adult worms were obtained from 73 patients before exposure to ivermectin and in the same patients following three years of annual or three-monthly treatment at 150 microg/kg or 800 microg/kg. Genotype frequencies were determined in beta-tubulin, a gene previously found to be linked to ivermectin selection and resistance in parasitic nematodes. Such frequencies were also determined in two other genes, heat shock protein 60 and acidic ribosomal protein, not known to be linked to ivermectin effects. In addition, we investigated the relationship between beta-tubulin genotype and female parasite fertility. We found a significant selection for beta-tubulin heterozygotes in female worms. There was no significant selection for the two other genes. Quarterly ivermectin treatment over three years reduced the frequency of the beta-tubulin "aa" homozygotes from 68.6% to 25.6%, while the "ab" heterozygotes increased from 20.9% to 69.2% in the female parasites. The female worms that were homozygous at the beta-tubulin locus were more fertile than the heterozygous female worms before treatment (67% versus 37%; p = 0.003) and twelve months after the last dose of ivermectin in the groups treated annually (60% versus 17%; p<0.001). Differences in fertility between heterozygous and homozygous worms were less apparent three months after the last treatment in the groups treated three-monthly. CONCLUSIONS: The results indicate that ivermectin is causing genetic selection on O. volvulus. This genetic selection is associated with a lower reproductive rate in the female parasites. We hypothesize that this genetic selection indicates that a population of O. volvulus, which is more tolerant to ivermectin, is being selected. This selection could have implications for the development of ivermectin resistance in O. volvulus and for the ongoing onchocerciasis control programmes.

Can the bis(diboranyl) structure of B(4)H(10) be observed? The story continues.

Pathways for the conversion of the unknown bis(diboranyl) isomer of tetraborane(10) (B(4)H(10)) to the known arachno isomer have been determined for the first time with the use of an electron correlation ab initio quantum chemical method and without the use of constraints in determination of the transition structures. Two isomers of tetraborane(10), one new, with a pentacoordinated boron atom have been found on the theoretical potential energy surface. Several other pathways for molecular rearrangement of tetraborane(10) have also been characterized. The theoretical method was MP2 theory with the 6-31G(d,p) basis set. The most likely pathway for the conversion of the bis(diboranyl) isomer of tetraborane(10) to the arachno isomer is a concerted pathway with two pentacoordinated intermediates. The highest energy transition state for this pathway lies 27.7 kcal/mol above the bis(diboranyl) isomer. At the same level of the theory, the bis(diboranyl) isomer lies 9.2 kcal/mol above the known arachno isomer. The two isomers with a pentacoordinated boron atom lie 12.5 and 13.1 kcal/mol above the arachno isomer.

Effects of standard and high doses of ivermectin on adult worms of Onchocerca volvulus: a randomised controlled trial.

BACKGROUND: At present, control of onchocerciasis depends almost entirely on yearly treatments with 150 microg/kg ivermectin. We aimed to compare the effect of higher doses, more frequent doses, or both with the standard regimen on adult Onchocerca volvulus. METHODS: We randomly allocated 657 patients who had onchocerciasis to 150 microg/kg ivermectin yearly (reference group), 150 microg/kg every 3 months, 400 then 800 microg/kg yearly, or 400 then 800 microg/kg every 3 months. We took skin snip samples from every patient before, and 3 years and 4 years after the first dose, and, at the same time excised one subcutaneous O volvulus nodule, which was examined histologically. The primary outcome was the vital status of the female worms. Analysis was done per protocol. FINDINGS: We obtained nodules from 511 patients. After 3 years of treatment, more female worms had died in the groups treated every 3 months than in the reference group (odds ratio=1.84 [95% CI 1.23-2.75], p=0.003 for 150 microg/kg; and 2.17 [1.42-3.31], p<0.001 for high doses). Female worms were also less fertile in these groups than in the reference group (0.24 [0.14-0.43], p<0.0001; and 0.14 [0.06-0.29], p<0.0001, respectively). No difference was recorded between groups treated yearly (p=0.83 for the proportion of dead females). Unexpected side-effects consisted of mild, temporary, subjective visual changes in patients on high-dose regimens. INTERPRETATION: Treatment with 3-monthly ivermectin could greatly reduce the number of female worms and acute itching and skin lesions; lower transmission of O volvulus; and change the duration of control programmes.