Potency variation of small-molecule chymase inhibitors across species

Chymases (EC 3.4.21.39) are mast cell serine proteinases that are variably expressed in different species and, in most cases, display either chymotryptic or elastolytic substrate specificity. Given that chymase inhibitors have emerged as potential therapeutic agents for treating various inflammatory, allergic, and cardiovascular disorders, it is important to understand interspecies differences of the enzymes as well as the behavior of inhibitors with them. We have expressed chymases from humans, macaques, dogs, sheep (MCP2 and MCP3), guinea pigs, and hamsters (HAM1 and HAM2) in baculovirus-infected insect cells. The enzymes were purified and characterized with kinetic constants by using chromogenic substrates. We evaluated in vitro the potency of five nonpeptide inhibitors, originally targeted against human chymase. The inhibitors exhibited remarkable cross-species variation of sensitivity, with the greatest potency observed against human and macaque chymases, with K_i values ranging from ∼0.4 to 72 nM. Compounds were 10-300-fold less potent, and in some instances ineffective, against chymases from the other species. The X-ray structure of one of the potent phosphinate inhibitors, JNJ-18054478, complexed with human chymase was solved at 1.8 Å resolution to further understand the binding mode. Subtle variations in the residues in the active site that are already known to influence chymase substrate specificity can also strongly affect the compound potency. The results are discussed in the context of selecting a suitable animal model to study compounds ultimately targeted for human chymase.     

Acute infections in children are accompanied by oxidative modification of LDL and decrease of HDL cholesterol, and are followed by thickening of carotid intima-media.

BACKGROUND: Atherosclerosis begins early in life. Infections might contribute to the pathogenesis of atherosclerosis. In this study, we investigated whether acute infections in children could alter the carotid wall morphology and the lipid profile. METHODS: Mean carotid intima-media thickness (IMT) was measured by high-resolution ultrasound in 28 hospitalised children (mean age: 5+/-2 years), who fulfilled the diagnostic criteria of acute infections (body temperature, >38 degrees C; C-reactive protein, >15mg/ml, and clinical), and in 20 age- and gender-matched controls. Antibodies against oxidised low-density lipoprotein (anti-oxLDL antibodies), as well as total and high-density lipoprotein cholesterol (HDL-C) were analysed in all children. The infection group was investigated both during the acute illness and 3 months after clinical recovery (post-infection). RESULTS: During the acute illness, the infection group had elevated anti-oxLDL antibodies and decreased HDL-C, as compared to those obtained at 3 months and in controls (p<0.05). These changes in the infection group were followed, at 3 months, by thickening of carotid intima-media. Those who received antibiotics during their acute illness had less carotid thickening than those who were not treated with antibiotics (p<0.05). CONCLUSION: Acute infections in children seem to be accompanied by enhanced oxidative modification of LDL and by decrease in HDL-C. These lipid changes may be followed by thickening of carotid artery intima-media. These findings suggest that, in childhood, acute infections could be associated with increased risk of atherosclerosis, and warrant further studies on this topic.     

Periodontal treatment needs in populations under 20 years of age in Espoo, Finland and Chiangmai, Thailand

The aim of this study was to assess the periodontal treatment needs at under 20 yr of age in the affluent area of Espoo, Finland, offering comprehensive public dental health care, as compared to a less advantaged area in Chiangmai, Thailand. In Espoo, 50 girls and 50 boys were examined in each age group of 7, 12 and 17 yr. In Chiangmai equal numbers of girls and boys were examined to obtain a group of 89 subjects aged 18.5 + 0.6 yr. According to the Community Periodontal Index of Treatment Needs (CPITN) the need of scaling increased in Espoo from 6% of the 7-yr-olds to 39% of the 17-yr-olds. Moderate pocketing (4-5 mm) occurred in one subject at age 12 and in three subjects at age 17. In Chiangmai, deep pockets (6 mm and over) were recorded for 1%, moderate pockets for a total of 44%, and dental calculus as the highest treatment need indicator in the remaining 55%, indicating a need for professional treatment in 100% of the group examined. The mean number of sextants requiring scaling was 0.6 per person at age 17 in Espoo as compared to 4.5 at 18.5 yr of age in Chiangmai. Three or more healthy sextants per subject were recorded for 47% of the 17-yr-olds in Espoo and for only 6% of the 18.5-yr-olds in Chiangmai. It was concluded that already at young age vast differences occur between periodontal treatment needs in industrialized and developing countries.     

Physical loading and performance as predictors of back pain in healthy adults A 5-year prospective study

We investigated muscle strength, aerobic power, and occupational and leisure-time physical loading as predictors of back pain in a 5-year follow-up study. A cohort of 456 adults aged 25, 35, 45 and 55 years, free of back pain, participated in measurements of anthropometric characteristics, aerobic power and muscle strength characteristics at baseline. The subjects' levels and types of physical activity and occupational physical loading were also determined. At 5 years after the baseline examinations 356 of these subjects (78.1 %) were reached by mail, and 262 of them (73.6%) properly completed and returned a questionnaire including a detailed back pain history for the 5 years following the baseline measurements. Of this number 56 subjects (21 %) who reported back pain ( > 30 on a scale from 0 to 100) and functional impairment during the 5-year follow-up composed the marked back pain group. Other subjects (n = 71, 27%) noting lesser symptoms were included in the mild back pain group; 135 subjects (52%) reported having had no back pain. The subjects with marked back pain were on average taller than the subjects without back pain, while no such difference was found in body mass. Heavy occupational musculoskeletal loading (P = 0.005) and high general occupational physical demands (P = 0.036) predicted future back pain. Leisuretime physical activity, aerobic power or muscle strength characteristics were not predictive of future back pain.     

Streptococcus pyogenes and Lactobacillus rhamnosus differentially induce maturation and production of Th1-type cytokines and chemokines in human monocyte-derived dendritic cells

Dendritic cells (DCs) are the most efficient antigen-presenting cells and thus, have a major role in regulating host immune responses. In the present study, we have analyzed the ability of Gram-positive, pathogenic Streptococcus pyogenes and nonpathogenic Lactobacillus rhamnosus to induce the maturation of human monocyte-derived DCs. Stimulation of DCs with S. pyogenes resulted in strong expression of DC costimulatory molecules CD80, CD83, and CD86 accompanied with a T helper cell type 1 (Th1) cytokine and chemokine response. S. pyogenes also induced interleukin (IL)-2 and IL-12 production at mRNA and protein levels. In addition, IL-23 and IL-27 subunits p40, p19, p28, and EBI3 were induced at mRNA level. In contrast, L. rhamnosus-stimulated DCs showed only moderate expression of costimulatory molecules and produced low levels of cytokines and chemokines. Furthermore, no production of IL-2 or IL-12 family cytokines was detected. Bacteria-induced DC maturation and especially cytokine and chemokine production were reduced when bacteria were heat-inactivated. Our results show that human monocyte-derived DCs respond differently to different Gram-positive bacteria. Although pathogenic S. pyogenes induced a strong Th1-type response, stimulation with nonpathogenic L. rhamnosus resulted in development of semi-mature DCs characterized by moderate expression of costimulatory molecules and low cytokine production.     

Vortex Shaped Current Sources in a Physical Torso Phantom

Recent studies reported differential information in human magnetocardiogram and in electrocardiogram. Vortex currents have been discussed as a possible source of this divergence. With the help of physical phantom experiments, we quantified the influence of active vortex currents on the strength of electric and magnetic signals, and we tested the ability of standard source localization algorithms to reconstruct vortex currents. The active vortex currents were modeled by a set of twelve single current dipoles arranged in a circle and mounted inside a phantom that resembles a human torso. Magnetic and electric data were recorded simultaneously while the dipoles were switched on stepwise one after the other. The magnetic signal strength increased continuously for an increasing number of dipoles switched on. The electric signal strength increased up to a semicircle and decreased thereafter. Source reconstruction with unconstrained focal source models performed well for a single dipole only (less than 3-mm localization error). Minimum norm source reconstruction yielded reasonable results only for a few of the dipole configurations. In conclusion active vortex currents might explain, at least in part, the difference between magnetically and electrically acquired data, but improved source models are required for their reconstruction.     

Haplotypic association of DDAH1 with susceptibility to pre-eclampsia

Association between pre-eclampsia (PEE1) and the dimethylarginine dimethylaminohydrolase (DDAH) 1 and 2 genes, which play a role in the regulation of nitric oxide synthesis and release, was studied. In a case-control study design single nucleotide polymorphisms (SNPs) were determined at eight sites in the DDAH1 gene and at one site (Pro231Pro) in the DDAH2 gene from 132 women with pre-eclampsia and 112 healthy controls. Three SNPs in the DDAH1 gene were associated with pre-eclampsia, showing complete linkage disequilibrium with each other, but none of the associations in the allele or genotype data reached statistical significance in either of the genes after the correction for multiple testing. Haplotype frequencies were estimated using a population based on a maximum likelihood method (EM algorithm). Four common DDAH1 haplotypes were present and a significant association of haplotypes H2 and H3 with pre-eclampsia (P=0.03) was found. The risk of pre-eclampsia was greatest in individuals (odds ratio: 3.93; 95% confidence interval: 1.54-9.99) who had two copies of the high-risk haplotypes (H2 or H3). The observed haplotypic association provides the first evidence of the importance of DDAH1 polymorphisms in pre-eclampsia susceptibility.     

Reelin-immunoreactivity in the hippocampal formation of 9-month-old wildtype mouse: effects of APP/PS1 genotype and ovariectomy

Reelin, an extracellular matrix protein has an important role in the migration, correct positioning and maturation of neurons during development. Though it is generally down-regulated in the postnatal period, expression of this large glycoprotein continues in the adult brain in some cell populations. In the present study, we examined the distribution of reelin-immunoreactivity (-ir) in the hippocampal formation of 9-month-old wildtype mice (WT). Then, reelin-ir in normal mice was compared to that of transgenic mice (APP/PS1) carrying mutated human APP and PS1 genes, which are linked to the familial form of Alzheimer's disease (AD). The APP/PS1 mice were additionally burdened with a second risk factor for AD, namely depletion of circulating gonadal hormones by ovariectomy (APP/PS1 + OVX). The analyses revealed that in adult WT reelin-ir is expressed by Cajal-Retzius cells and a subgroup of interneurons throughout the hippocampal formation. In addition, layer II projection neurons in the lateral entorhinal subfields are reelin-ir. Interestingly, ovariectomy decreases the number of reelin-ir cells in the hilus in WT mice, whereas AD-related genotype alone induces only a non-significant reduction. Unexpectedly, additional stress, e.g., depletion of gonadal hormones, does not aggravate the slight reduction in the reelin cell number in the APP/PS1 mice. We propose that the changes in normal reelin-ir are linked to disturbances in repair mechanisms in which APP/PS1 and gonadal hormones are involved and which are perturbed in neurodegenerative conditions, namely AD.