RTS,S malaria vaccine efficacy and immunogenicity during Plasmodium falciparum challenge is associated with HLA genotype.

Although RTS,S remains the most advanced malaria vaccine, the factors influencing differences in vaccine immunogenicity or efficacy between individuals or populations are still poorly characterised. The analyses of genetic determinants of immunogenicity have previously been restricted by relatively small sample sizes from individual trials. Here we combine data from six Phase II RTS,S trials and evaluate the relationship between HLA allele groups and RTS,S-mediated protection in controlled human malaria infections (CHMI), using multivariate logistic or linear regression. We observed significant associations between three allele groups (HLA-A∗01, HLA-B∗08, and HLA-DRB1∗15/∗16) and protection, while another three allele groups (HLA-A∗03, HLA-B∗53, and HLA-DRB1∗07) were significantly associated with lack of protection. It is noteworthy that these 'protective' allele groups are thought to be at a lower prevalence in sub-Saharan African populations than in the UK or USA where these Phase II trials occurred. Taken together, the analyses presented here give an indication that HLA genotype may influence RTS,S-mediated protective efficacy against malaria infection.

[The malaria vaccine candidate RTS,S/AS is in phase III clinical trials]. / Le candidat vaccin antipaludique RTS,S/AS est entré en essais cliniques de phase III.

This review paper describes the development of the RTS,S/AS vaccine, from concept to phase III testing. The rationale for selection of the circumsporozoite protein (CSP) as the target antigen and the preclinical development history of the vaccine are described. The RTS,S/AS candidate vaccine has been evaluated in multiple phase I/II studies and was shown to have a favorable safety profile and to be well tolerated in both adults and children. Consistent and significant efficacy has been observed in the target population of infants and children against Plasmodium falciparum infection and disease in different transmission settings, in different age groups, with or without Expanded Program of Immunization (EPI) vaccine co-administration. The RTS,S/AS01(E) malaria vaccine candidate has recently entered phase III testing. Reaching this important milestone is the culmination of more than 20 years of research and development by GlaxoSmithKline, their partners and collaborators. If the phase III results confirm the observations made during phase II testing, the RTS,S/AS01(E) vaccine, when broadly implemented and judiciously integrated with other malaria-prevention measures, would have a major public-health impact in sub-Saharan Africa.

Mapping slow waves and spikes in chronically instrumented conscious dogs: implantation techniques and recordings.

Myoelectric recordings from the intestines in conscious animals have been limited to a few electrode sites with relatively large inter-electrode distances. The aim of this project was to increase the number of recording sites to allow high-resolution reconstruction of the propagation of myoelectrical signals. Sets of six unipolar electrodes, positioned in a 3x2 array, were constructed. A silver ring close to each set served as the reference electrodes. Inter-electrode distances varied from 4 to 8 mm. Electrode sets, to a maximum of 4, were implanted in various configurations allowing recording from 24 sites simultaneously. Four sets of 6 electrodes each were implanted successfully in 11 female Beagles. Implantation sites evaluated were the upper small intestine (n=10), the lower small intestine (n=4) and the stomach (n=3). The implants remained functional for 7.2 months (median; range 1.4-27.3 months). Recorded signals showed slow waves at regular intervals and spike potentials. In addition, when the sets were positioned close together, it was possible to re-construct the propagation of individual slow waves, to determine their direction of propagation and to calculate their propagation velocity. No signs or symptoms of interference with normal GI-function were observed in the tested animals. With this approach, it is possible to implant 24 extracellular electrodes on the serosal surface of the intestines without interfering with its normal physiology. This approach makes it possible to study the electrical activities of the GI system at high resolution in vivo in the conscious animal.

Respuesta inmune en neonatos no infectados nacidos de madres infectadas por Trypanosoma cruzi / Immune responses of non-infected neonates of mothers infected with Trypanosoma cruzi

We have investigated if maternal T. cruzi infection could induce in utero innate and/or adaptive immune responses in uninfected neonates by measuring specific IgM and IgA antibodies in cord blood plasma, and by performing phenotypic and functional studies of umbilical cord blood cells of their newborns (M+B- group). We detected T. cruzi-specific IgM and IgA antibodies in M+B- cord blood, indicating they had mounted in utero a strong B cell response, although they are not infected. On the other hand, circulating T cells of such uninfected neonates displayed a low level of activation, as seen bya slightly increased expression of the activation markers CD45RO on CD4+ T cells and HLA-DR on CD8+ T cells, although the proportion of CD4+ and CD8+ T cells was unmodified as compared to newborns from uninfected mothers (MB- group). This activation did not give rise to a proliferative response upon stimulation by T. cruzi antigens in vitro. However, M+B- cells produced low levels of lymphokines (IFN-gamma and IL-13) upon mitogenic stimulation, which was not the case of M-B- newborn cells. Beside this, M+B- blood cells produced higher levels of inflammatory cytokines (IL-1b, IL-6, TNF-alpha) than M-B- cells when stimulated with the T. cruzi lysate or LPS, suggesting the over-activation of the innate response in M+B- newborns. Monocytes participated in such inflammatory response since M+B- purified cord blood monocytes produced higher levels of TNF- when incubated with LPS or a T. cruzi lysate than M-B- cells. Altogether, these results show that, even in the absence of congenital infection, maternal T. cruzi infection triggers in utero both adaptive and innate immune responses in their babies. This indicates that parasite circulating antigens have been transferred from mothers to their fetuses.

T cell responses to vaccines in infants: defective IFNgamma production after oral polio vaccination.

The immaturity of the neonatal immune system is associated with an increased susceptibility to infections. Studies in mice indicate that neonatal immune responses are biased towards the T helper 2 type, but little is known about helper T cell responses in human newborns. In this study, the oral polio vaccine was used as a model of early immunization to investigate the capacity of young infants to develop cellular immune responses. We show that neonatal immunization with oral polio vaccine induces the production of high titres of neutralizing antibodies but reduced proliferative and IFNgamma responses to polio antigens compared to immune adults. These data suggest that specific strategies will be required to immunize newborns against pathogens controlled by Th1 type immune responses.

Hierarchical formation of helical supramolecular polymers via stacking of hydrogen-bonded pairs in water.

Bifunctional ureido-s-triazines provided with penta(ethylene oxide) side chains are able to self assemble in water, leading to helical columns via cooperative stacking of the hydrogen-bonded pairs (DADA array). Monofunctional ureido-s-triazines do not form such helical architectures. The presence of a linker, covalently connecting the two ureido-s-triazine units, is essential as it generates a high local concentration of aromatic units, favorable for stacking interactions. This hydrophobic stacking of the aromatic units occurs at concentrations as low as 5 x 10(-6) M and can be visualized by using fluorescence spectroscopy. The stacking generates a hydrophobic microenvironment that allows intermolecular hydrogen bonding to occur at higher concentrations because the hydrogen bonds are shielded from competitive hydrogen bonding with water. This hierarchical process results in the formation of a helical self-assembled polymer in water at concentrations above 10(-4) M. Chiral side chains attached to the ureido-s-triazine units bias the helicity of these columns as concluded from CD spectroscopy and "Sergeants and Soldiers" experiments.

Tuberculosis contacts but not patients have higher gamma interferon responses to ESAT-6 than do community controls in The Gambia.

The Mycobacterium tuberculosis antigen ESAT-6 has been proposed for tuberculosis immunodiagnosis. In The Gambia, 30% of community controls produced gamma interferon (IFN-gamma) in response to ESAT-6. Increased proportions of responders and intensities of responses were found in household contacts. Responses that were initially low in tuberculosis patients increased after treatment. An ESAT-6 IFN-gamma assay will be of limited use in the diagnosis of tuberculosis in countries where tuberculosis is endemic. Its role in contact tracing should be evaluated further.

Neonatal bacillus Calmette-Guérin vaccination induces adult-like IFN-gamma production by CD4+ T lymphocytes.

The immaturity of the neonatal immune system in mice is associated with defective IFN-gamma production and Th2-biased immune responses. In this study, infants vaccinated at birth with BCG produced similar concentrations of IFN-gamma in response to PPD and showed similar frequencies of IFN-gamma-producing lymphocytes as compared to immune adults. Infants and adults produced only low concentrations of IL-4 and IL-5. CD4+ T lymphocytes were the main source of IFN-gamma. Similar proportions of Th1 and Th0 PPD-specific T cell clones were observed in infants and adults. This study demonstrates that the human neonatal immune response to BCG is not biased towards Th2 and is characterized by the predominant production of IFN-gamma by CD4+ T lymphocytes.

HLA-B*35-restricted CD8 T cell epitopes in the antigen 85 complex of Mycobacterium tuberculosis.

Few target epitopes have been described for human CD8 T lymphocytes in antigens of Mycobacterium tuberculosis. By use of a reverse immunogenetics approach, 23 motif-bearing peptides of the Ag85 complex were tested for binding to HLA-B*35, one of the common B-types in West Africa. Three 9-mer peptides bound with high affinity to HLA-B*3501 and displayed low dissociation rates of peptide-major histocompatibility complexes (MHCs). IC(50) and half-life values of peptide-MHC class I complexes were in the same range as reported earlier for other immunogenic peptides. Immune responses against peptide Ag85C (aa 204-212) WPTLIGLAM were characterized in detail. Peptide-stimulated effector cells were able to kill macrophages infected with M. tuberculosis or bacille Calmette-Guérin. Peptide-specific CD8 T cells could be visualized by using HLA-B*3501 tetramers and were shown to produce interferon-gamma and tumor necrosis factor-alpha. Together with other published epitopes, these peptides can be used to study more closely the role of CD8 T cells in mycobacterial infection and tuberculosis.

Polarization of PPD-specific T-cell response of patients with tuberculosis from Th0 to Th1 profile after successful antimycobacterial therapy or in vitro conditioning with interferon-alpha or interleukin-12.

The T helper (Th) 1/Th2 balance in the T-lymphocyte response to purified protein derivative (PPD) was evaluated at the clonal level in six Italian and five Gambian patients with pulmonary tuberculosis (TB) before and after antimycobacterial therapy, as well as in five Gambian and four Italian healthy immune control subjects. In untreated patients, most PPD-specific clones derived from either peripheral blood or pleural effusions showed a Th0 cytokine profile (production of both interferon [IFN]-gamma and interleukin [IL]-4/IL-5). After 6 mo of therapy and clinical healing, most PPD-specific clones showed a polarized Th1 profile (production of IFN-gamma but not IL-4/IL-5) in both Italian and Gambian patients. The Th1 polarization was less marked in Gambian than in Italian patients and failed to occur in another group of four Italian patients who experienced treatment failure. The cytokine profile observed after successful therapy in patients with TB was similar to that found in healthy control subjects. T-cell clones of undefined specificity generated from PPD-stimulated cultures showed a similar Th0/Th2 bias in Gambian individuals and Italian patients with treatment failure. The Th0/Th2-biased responses in Gambian patients before therapy could be modulated in vitro by IFN-alpha or IL-12, which induced a Th1 polarization of both PPD-specific and bystander T cells. Our data show that active TB associates with a predominant Th0 response to mycobacterial antigens that could play a role in the pathogenesis of the disease. Adjunctive immunotherapy using Th1-polarizing cytokines could increase host defense against mycobacteria and accelerate healing.

Deficient IL-12(p35) gene expression by dendritic cells derived from neonatal monocytes.

To gain insight into the defects responsible for impaired Th1 responses in human newborns, we analyzed the production of cytokines by dendritic cells (DC) derived from cord blood monocytes. We observed that neonatal DC generated from adherent cord blood mononuclear cells cultured for 6 days in the presence of IL-4 and GM-CSF show a phenotype similar to adult DC generated from adherent PBMC, although they express lower levels of HLA-DR, CD80, and CD40. Measurement of cytokine levels produced by neonatal DC upon stimulation by LPS, CD40 ligation, or poly(I:C) indicated a selective defect in the synthesis of IL-12. Determination of IL-12(p40) and IL-12(p35) mRNA levels by real-time RT-PCR revealed that IL-12(p35) gene expression is highly repressed in stimulated neonatal DC whereas their IL-12(p40) gene expression is not altered. The addition of rIFN-gamma to LPS-stimulated newborn DC restored their expression of IL-12(p35) and their synthesis of IL-12 (p70) up to adult levels. Moreover, we observed that neonatal DC are less efficient than adult DC to induce IFN-gamma production by allogenic adult CD4(+) T cells. This defect was corrected by the addition of rIL-12. We conclude that neonatal DC are characterized by a severe defect in IL-12(p35) gene expression which is responsible for an impaired ability to elicit IFN-gamma production by T cells.

Peroxynitrite scavenging of flavonoids: structure activity relationship.

Peroxynitrite can oxidise and nitrosylate biomolecules and is associated with several diseases. The peroxynitrite scavenging of substituted phenols and several flavonoids was studied. The activity of phenol (poor scavenger) is positively influenced by electron donating substituents. A good correlation was found between the peroxynitrite scavenging activity of the substituted phenols and the Hammett σ or the E(HOMO). Flavonols containing a catechol group (3'- and 4'-OH) in ring B (rutin and monohydroxyethyl rutoside) or an AC-ring with three OH groups (3-, 5- and 7-OH) were potent scavengers. Evidence has been produced that in the AC-ring the 3-OH group was the reactive centre and that the reactivity of this group was positively influenced by electron donating groups at the 5 and/or 7 position (galangin, kaempferol, trihydroxyethyl quercetin).

Helical self-assembled polymers from cooperative stacking of hydrogen-bonded pairs.

The double helix of DNA epitomizes this molecule's ability to self-assemble in aqueous solutions into a complex chiral structure using hydrogen bonding and hydrophobic interactions. Non-covalently interacting molecules in organic solvents are used to design systems that similarly form controlled architectures. Peripheral chiral centres in assemblies and chiral side chains attached to a polymer backbone, have been shown to induce chirality at the supramolecular level, and highly ordered structures stable in water are also known. However, it remains difficult to rationally exploit non-covalent interactions for the formation of chiral assemblies that are stable in water, where solvent molecules can compete effectively for hydrogen bonds. Here we describe a general strategy for the design of functionalized monomer units and their association in either water or alkanes into non-covalently linked polymeric structures with controlled helicity and chain length. The monomers consist of bifunctionalized ureidotriazine units connected by a spacer and carrying solubilizing chains at the periphery. This design allows for dimerization through self-complementary quadruple hydrogen bonding between the units and solvophobically induced stacking of the dimers into columnar polymeric architectures, whose structure and helicity can be adjusted by tuning the nature of the solubilizing side chains.

Maternal Trypanosoma cruzi infection upregulates capacity of uninfected neonate cells To produce pro- and anti-inflammatory cytokines.

The possibility of maternal in utero modulation of the innate and/or adaptive immune responses of uninfected newborns from Trypanosoma cruzi-infected mothers was investigated by studying the capacity of their whole blood cells to produce cytokines in response to T. cruzi lysate or lipopolysaccharide-plus-phytohemagglutinin (LPS-PHA) stimulation. Cells of such newborns occasionally released gamma interferon (IFN-gamma) and no interleukin-2 (IL-2) and IL-4 upon specific stimulation, while their mothers responded by the production of IFN-gamma, IL-2, and IL-4. Infection in mothers was also associated with a hyperactivation of maternal cells and also, strikingly, of cells of their uninfected neonates, since their release of proinflammatory (IL-1beta, IL-6, and tumor necrosis factor alpha [TNF-alpha]) as well as of anti-inflammatory (IL-10 and soluble TNF receptor) cytokines or factors was upregulated in the presence of LPS-PHA and/or parasite lysate. These results show that T. cruzi infection in mothers induces profound perturbations in the cytokine response of their uninfected neonates. Such maternal influence on neonatal innate immunity might contribute to limit the occurrence and severity of congenital infection.

Ladderlike oligomers; intramolecular hydrogen bonding, push-pull character, and electron affinity.

Symmetrical 2,5-bis(2-aminophenyl)pyrazines have been synthesized by application of the Stille coupling strategy. These cotrimers feature three important properties, namely strong intramolecular hydrogen bonding, push-pull character, and high electron affinity. The presence of intramolecular hydrogen bonds has been confirmed by 1H NMR, IR spectroscopy, and single-crystal X-ray diffraction. The hydrogen bond strength can be increased by substituting the amino groups with stronger electron-withdrawing functionalities. Despite the anticipated enhanced pi-conjugation through planarization, a hypsochromic shift was observed in the UV/Vis spectra, explained by a decrease in push-pull character. The electron affinity of the cotrimers was deduced from the first reduction potentials measured by cyclic voltammetry and is related to the electron-withdrawing character of the amino substituents. The results obtained have been compared with those of the corresponding 4-aminophenyl analogues and show that intramolecular hydrogen bonds can be used to design polymers with enhanced pi conjugation as well as a high electron affinity.

Synthesis, estrogen receptor binding, and tissue distribution of a new iodovinylestradiol derivative (17alpha,20E)-21-[123I]Iodo-11beta-nitrato-19-norp regna-1,3,5 (10),20-tetraene-3,17-diol (E-[123I]NIVE).

We have synthesized and evaluated E-11beta-nitrato-17alpha-iodovinylestradiol (E-NIVE; E-3c) and its 123I-labelled form, as a new potential radioligand for imaging of estrogen receptor (ER)-positive human breast tumors. E-[123I]NIVE was prepared by stereospecific iododestannylation of the E-tri-n-butylstannylvinyl precursor (E-2c), obtained from reaction of 11beta-nitrato-estrone (8) with E-tributylstannylvinyllithium. In competitive binding studies, E-NIVE proved to have high binding affinity for both the rat and the human ER (Ki 280-730 pM), without significant binding to human sex hormone binding globulin. Distribution studies in normal and mammary tumor-bearing rats showed specific ER-mediated uptake of E-[123I]NIVE in the estrogen target tissues, i.e., uterus, ovaries, pituitary, and hypothalamus, but not in the mammary tumors. Selective retention in these target tissues, including tumor tissue, resulted in significant increases over time for the target tissue-to-muscle uptake ratios, but not for the target tissue-to-fat uptake ratios. The tumor-to-fat uptake ratio even appeared constantly below 1. In the primary estrogen target tissues, E-[123I]NIVE displayed high specific ER-mediated uptake and retention, which resulted in moderate target-to-nontarget tissue uptake ratios. In contrast, in tumor tissue, E-[123I]NIVE uptake appeared to be rather low and not ER-specific. As a consequence, E-[123I]NIVE appears to be a less favorable radioligand for ER imaging in breast cancer than the previously studied stereoisomers of 11beta-methoxy-17alpha-[123I]iodovinylestradiol (E- and Z-[123I]MIVE; [123I]E- and [123I]Z-3b).

Development of radioligands for the imaging of cardiac beta-adrenoceptors using SPECT. Part I: Asymmetric synthesis and structural characterization of five new iodine-containing beta-adrenoceptor antagonist derivatives.

The asymmetric synthesis of a series of iodinated beta-adrenoceptor ligands is described. One ligand, (S)-(-)-[1-(2-iodophenoxy)]-3'-(tert-butylamino)-2'-propanol (CYBL3), is based on the beta-adrenoceptor antagonist penbutolol. The other ligands are N-iodovinyl and N-iodoaryl analogues of the beta-adrenoceptor antagonist CGP12177. These have been synthesized from 2-amino-3-nitrophenol. Furthermore, radioiodinated [123I]CYBL3 and [123I](2'S,2"E)-[4-(3'-(1",1"-dimethyl-3"-iodo-2" propenylamino)-2'-hydroxy propoxy)]-benzimidazol-2-one have been prepared by radiolabelling the corresponding trialkyltin precursors using [123I]-NaI in the presence of hydrogen peroxide.

Extended-Core Discotic Liquid Crystals Based on the Intramolecular H-Bonding in N-Acylated 2,2'-Bipyridine-3,3'-diamine Moieties.

A new type of disc-shaped molecule (1 a-c) has been synthesised and characterised. The molecules were built up by linking three lipophilic, N-monoacylated 2,2'-bipyridine-3,3'-diamine wedges to a central 1,3,5-benzenetricarbonyl unit. They show liquid crystalline behaviour, as shown by DSC, polarisation microscopy and X-ray diffraction. In all cases the mesophase was characterised as a Dho phase. From (1) H NMR results it was shown that the interior of compounds 1 a-c preferentially adopts a C3 symmetrical conformation owing to strong intramolecular H-bonding, which gives rise to an extended core. This large core induces strong interactions between molecules, leading to mesophases of enhanced thermal stability.

Synthesis and in vitro and in vivo characteristics of an iodinated analogue of the beta-adrenoceptor antagonist carazolol.

A new (radio)iodinated, beta-adrenoceptor ligand, (S)-(-)-4-[3-[(1,1-dimethyl-3-iodo-(2E)-propenyl)-amino]-2- hydroxypropoxy]carbazole (CYBL8E, 1), was prepared. 1 is an iodinated analogue of the high-affinity beta-adrenoceptor antagonist carazolol (2). The asymmetric synthesis was achieved in four steps starting from 4-hydroxycarbazole. The iodine-123-labeled form was obtained by an iododestannylation reaction with [123I]NaI in the presence of H2O2. Using classical in vitro displacement experiments with membrane fractions of cardiac left ventricular muscle, 1 proved to have a high affinity for the receptor (Ki = 0.31 +/- 0.03). Biodistribution studies performed in New Zealand white rabbits demonstrated the specificity of the binding in vivo to the receptor. Uptake of [123I]1 was reduced significantly in both atrial muscle, left ventricular muscle, frontal cortex, cerebellum, and striatum, by the pretreatment of the animals with different beta-adrenoceptor antagonists. In conclusion, 1 is a potent nonselective beta-adrenoceptor antagonist, which binds specifically to the beta-adrenoceptor in vivo, and is therefore a promising radioligand for the imaging of beta-adrenoceptors using single photon emission computerized tomography.