Allelic variation in HLA-B and HLA-C sequences and the evolution of the HLA-B alleles

Several new HLA-B (B8, B51, Bw62)- and HLA-C (Cw6, Cw7)-specific genes were isolated either as genomic cosmid or cDNA clones to study the diversity of HLA antigens. The allele specificities were identified by sequence analysis in comparison with published HLA-B and -C sequences, by transfection experiments, and Southern and northern blot analysis using oligonucleotide probes. Comparison of the classical HLA-A, -B, and -C sequences reveals that allele-specific substitutions seem to be rare events. HLA-B51 codes only for one allelespecific residue: arginine at position 81 located on the 1 helix, pointing toward the antigen binding site. HLA-B8 contains an acidic substitution in amino acid position 9 on the first central sheet which might affect antigen binding capacity, perhaps in combination with the rare replacement at position 67 (F) on the ul helix. HLA-B8 shows greatest homology to HLA-Bw42, -Bw41, -B7, and-Bw60 antigens, all of which lack the conserved restriction sites Pst I at position 180 and Sac I at position 131. Both sites associated with amino acid replacements seem to be genetic markers of an evolutionary split of the HLA-B alleles, which is also observed in the leader sequences. HLA-Cw7 shows 98% sequence identity to the JY328 gene. In general, the HLA-C alleles display lower levels of variability in the highly polymorphic regions of the 1 and 2 domains, and have more distinct patterns of locus-specific residues in the transmembrane and cytoplasmic domains. Thus we propose a more recent origin for the HLA-C locus.     

HCMV-infection in a human arterial organ culture model: effects on cell proliferation and neointimal hyperplasia

Background

The impact of infections with the human cytomegalovirus (HCMV) for the development of atherosclerosis and restenosis is still unclear. Both a clear correlation and no correlation at all have been reported in clinical, mostly serological studies. In our study we employed a human non-injury ex vivo organ culture model to investigate the effect of an in vitro permissive HCMV-infection on cell proliferation and neointimal hyperplasia for a period of 56 days.

Results

During routine-nephrectomies parts of renal arteries from 71 patients were obtained and prepared as human organ cultures. Cell free HCMV infection was performed with the fibroblast adapted HCMV strain AD169, the endotheliotropic strain TB40E, and a clinical isolate (AN 365). After 3, 7, 14, 21, 28, 35, and 56 days in culture staining of HCMV-antigens was carried out and reactive cell proliferation and neointimal thickening were analysed. Successful HCMV-infection was accomplished with all three virus strains studied. During the first 21 days in organ culture no cell proliferation or neointimal hyperplasia was detected. At day 35 and day 56 moderate cell proliferation and neointimal hyperplasia was found both in HCMV-infected segments and mock infected controls. Neointimal hyperplasia in productively HCMV-infected segments was lower than in non infected at day 35 and day 56, but relatively higher after infection with the endotheliotropic TB40E in comparison with the two other strains.

Conclusion

The data do not support the hypothesis that HCMV-infection triggers restenosis via a stimulatory effect on cell proliferation and neointimal hyperplasia in comparison to non infected controls. Interestingly however, even after lytic infection, a virus strain specific difference was observed.     

Masitinib (AB1010), a Potent and Selective Tyrosine Kinase Inhibitor Targeting KIT

Background

The stem cell factor receptor, KIT, is a target for the treatment of cancer, mastocytosis, and inflammatory diseases. Here, we characterise the in vitro and in vivo profiles of masitinib (AB1010), a novel phenylaminothiazole-type tyrosine kinase inhibitor that targets KIT.

Methodology/Principal Findings

In vitro, masitinib had greater activity and selectivity against KIT than imatinib, inhibiting recombinant human wild-type KIT with an half inhibitory concentration (IC₅₀) of 200±40 nM and blocking stem cell factor-induced proliferation and KIT tyrosine phosphorylation with an IC₅₀ of 150±80 nM in Ba/F3 cells expressing human or mouse wild-type KIT. Masitinib also potently inhibited recombinant PDGFR and the intracellular kinase Lyn, and to a lesser extent, fibroblast growth factor receptor 3. In contrast, masitinib demonstrated weak inhibition of ABL and c-Fms and was inactive against a variety of other tyrosine and serine/threonine kinases. This highly selective nature of masitinib suggests that it will exhibit a better safety profile than other tyrosine kinase inhibitors; indeed, masitinib-induced cardiotoxicity or genotoxicity has not been observed in animal studies. Molecular modelling and kinetic analysis suggest a different mode of binding than imatinib, and masitinib more strongly inhibited degranulation, cytokine production, and bone marrow mast cell migration than imatinib. Furthermore, masitinib potently inhibited human and murine KIT with activating mutations in the juxtamembrane domain. In vivo, masitinib blocked tumour growth in mice with subcutaneous grafts of Ba/F3 cells expressing a juxtamembrane KIT mutant.

Conclusions

Masitinib is a potent and selective tyrosine kinase inhibitor targeting KIT that is active, orally bioavailable in vivo, and has low toxicity.     

Evolution and population structure of Salmonella enterica serovar Newport

Salmonellosis caused by Salmonella enterica serovar Newport is a major global public health concern, particularly because S. Newport isolates that are resistant to multiple drugs (MDR), including third-generation cephalosporins (MDR-AmpC phenotype), have been commonly isolated from food animals. We analyzed 384 S. Newport isolates from various sources by a multilocus sequence typing (MLST) scheme to study the evolution and population structure of the serovar. These were compared to the population structure of S. enterica serovars Enteritidis, Kentucky, Paratyphi B, and Typhimurium. Our S. Newport collection fell into three lineages, Newport-I, Newport-II, and Newport-III, each of which contained multiple sequence types (STs). Newport-I has only a few STs, unlike Newport-II or Newport-III, and has possibly emerged recently. Newport-I is more prevalent among humans in Europe than in North America, whereas Newport-II is preferentially associated with animals. Two STs of Newport-II encompassed all MDR-AmpC isolates, suggesting recent global spread after the acquisition of the bla(CMY-2) gene. In contrast, most Newport-III isolates were from humans in North America and were pansusceptible to antibiotics. Newport was intermediate in population structure to the other serovars, which varied from a single monophyletic lineage in S. Enteritidis or S. Typhimurium to four discrete lineages within S. Paratyphi B. Both mutation and homologous recombination are responsible for diversification within each of these lineages, but the relative frequencies differed with the lineage. We conclude that serovars of S. enterica provide a variety of different population structures.     

Role of Carboxydobacteria in Consumption of Atmospheric Carbon Monoxide by Soil

The carbon monoxide consumption rates of the carboxydobacteria Pseudomonas (Seliberia) carboxydohydrogena, P. carboxydovorans, and P. carboxydoflava were measured at high (50%) and low (0.5 μl liter⁻¹) mixing ratios of CO in air. CO was only consumed when the bacteria had been grown under CO-autotrophic conditions. As an exception, P. carboxydoflava consumed CO also after heterotrophic growth on pyruvate. At low cell densities the CO consumption rates measured at low CO mixing ratios were similar in cell suspensions and in mixtures of bacteria in soil. CO consumption observed in natural soil (loess, eolian sand, chernozem) as well as in suspensions or soil mixtures of carboxydobacteria showed Michaelis-Menten kinetics. The K_m values for CO of the carboxydobacteria (K_m = 465 to 1,110 μl of CO liter⁻¹) were much higher than those of the natural soils (K_m = 5 to 8 μl of CO liter⁻¹). Considering the difference of the K_m values and the observed V_max values, carboxydobacteria cannot contribute significantly to the consumption of atmospheric CO.     

18F]Galacto-RGD: synthesis, radiolabeling, metabolic stability, and radiation dose estimates

It has been demonstrated in various murine tumor models that radiolabeled RGD-peptides can be used for noninvasive determination of alphavbeta3 integrin expression. Introduction of sugar moieties improved the pharmacokinetic properties of these peptides and led to tracer with good tumor-to-background ratios. Here we describe the synthesis, radiolabeling, and the metabolic stability of a glycosylated RGD-peptide ([18F]Galacto-RGD) and give first radiation dose estimates for this tracer. The peptide was assembled on a solid support using Fmoc-protocols and cyclized under high dilution conditions. It was conjugated with a sugar amino acid, which can be synthesized via a four-step synthesis starting from pentaacetyl-protected galactose. For radiolabeling of the glycopeptide, 4-nitrophenyl-2-[18F]fluoropropionate was used. This prosthetic group allowed synthesis of [18F]Galacto-RGD with a maximum decay-corrected radiochemical yield of up to 85% and radiochemical purity >98%. The overall radiochemical yield was 29 +/- 5% with a total reaction time including final HPLC preparation of 200 +/- 18 min. The metabolic stability of [18F]Galacto-RGD was determined in mouse blood and liver, kidney, and tumor homogenates 2 h after tracer injection. The average fraction of intact tracer in these organs was approximately 87%, 76%, 69%, and 87%, respectively, indicating high in vivo stability of the radiolabeled glycopeptide. The expected radiation dose to humans after injection of [18F]Galacto-RGD has been estimated on the basis of dynamic PET studies with New Zealand white rabbits. According to the residence times in these animals the effective dose was calculated using the MIRDOSE 3.0 program as 2.2 x 10(-2) mGy/MBq. In conclusion, [18F]Galacto-RGD can be synthesized in high radiochemical yields and radiochemical purity. Despite the time-consuming synthesis of the prosthetic group 185 MBq of [18F]Galacto-RGD, a sufficient dose for patient studies, can be produced starting with approximately 2.2 GBq of [18F]flouride. Moreover, the fast excretion, the suitable metabolic stability and the low estimated radiation dose allow to evaluate this tracer in human studies.     

Specificity of IgG and IgE antibodies against plant and insect glycoprotein glycans determined with artificial glycoforms of human transferrin

Cross-reactive carbohydrate determinants of plants are essentially a mixture of N-glycans containing beta1,2-xylose and core alpha1,3-fucose, the latter also found in insect glycoproteins. To determine the relative contributions of these two sugar residues to antibody binding, we prepared an array of glycomodified forms of human apo-transferrin. Using core-alpha1, 3-fucosyltransferase (EC 2.4.1.214) and beta1,2-xylosyltransferase (EC 2.4.2.38) recombinantly expressed in Pichia pastoris and suitable glycosidases, glycoforms containing either only fucose (MMF), only xylose (MMX), both (MMXF), or neither (MM) linked to the common pentasaccharide core were generated. Additional glycoforms were obtained by enzymatic removal of the alpha1,3-linked mannosyl residue. These transferrin glycoforms served to define the binding specificity of antibodies in western blot, ELISA, and inhibition ELISA. Rabbit anti-horseradish peroxidase serum bound to both the fucosylated (MMF) and the xylosylated (MMX) glycoforms. Inhibition studies indicated two independent highly specific populations reacting with either of the two epitopes. In contrast, the monoclonal antibody YZ1/2.23 appears to recognize a larger structure including both the fucosyl and the xylosyl residue. The mannose-deficient glycoform was a poorer inhibitor for both antibodies. Terminal GlcNAc residues prevented antibody binding. Rabbit anti-bee venom serum reacted with fucosylated forms (MMF and MMXF) only. Experiments with sera from allergic patients suggest that glycomodified human transferrin, especially the MMXF glycoform, is a suitable reagent for the detection of antibodies against cross-reactive carbohydrate determinants. Within the panel studied, several sera contained high levels of fucose-reactive IgE but only a few sera showed any binding to MMX-transferrin.     

Effect of five tree crops and a cover crop in multi-strata agroforestry at two fertilization levels on soil fertility and soil solution chemistry in central Amazonia

The spatio-temporal patterns of soil fertility and soil solution chemistry in a multi-strata agroforestry system with perennial crops were analysed as indicators for the effects of crop species and management measures on soil conditions under permanent agriculture in central Amazonia. The study was carried out in a plantation with locally important tree crop species and a leguminous cover crop at two fertilization levels on a xanthic Ferralsol. Soil fertility to 2 m soil depth was evaluated 3.5 years after the establishment of the plantation, and soil solution chemistry at 10, 60 and 200 cm soil depth was monitored over 2 years. Several soil fertility characteristics exhibited spatial patterns within the multi-strata plots which reflected the differing properties of the plant species and their management, including the fertilizer input. Significant differences between species could be detected to 150 cm depth, and between fertilization treatments to 200 cm depth. Favourable effects on nutrient availability in the soil were found for annatto (Bixa orellana) (P, K) and cupuaçu (Theobroma grandiflorum) (Ca, Mg) in comparison with peach palm (Bactris gasipaes) and Brazil nut (Bertholletia excelsa). Nutrient concentrations of the soil solution showed pronounced fluctuations in the topsoil, corresponding to fertilizer applications. Large nutrient concentrations in the soil solution were accompanied by increased concentrations of aluminium and low pH values, caused by exchange reactions between fertilizer and sorbed acidity and reinforced by the acidifying effect of nitrification. The soil solution under the leguminous cover crop Pueraria phaseoloides had relatively large N concentrations during periods when those under the tree crops were small, and this could partly explain why no yield responses to N fertilization were observed at this site.