Developmental determinants in non-communicable chronic diseases and ageing.

Prenatal and peri-natal events play a fundamental role in health, development of diseases and ageing (Developmental Origins of Health and Disease (DOHaD)). Research on the determinants of active and healthy ageing is a priority to: (i) inform strategies for reducing societal and individual costs of an ageing population and (ii) develop effective novel prevention strategies. It is important to compare the trajectories of respiratory diseases with those of other chronic diseases.

Thick collagen-based 3D matrices including growth factors to induce neurite outgrowth.

Designing synthetic microenvironments for cellular investigations is a very active area of research at the crossroads of cell biology and materials science. The present work describes the design and functionalization of a three-dimensional (3D) culture support dedicated to the study of neurite outgrowth from neural cells. It is based on a dense self-assembled collagen matrix stabilized by 100-nm-wide interconnected native fibrils without chemical crosslinking. The matrices were made suitable for cell manipulation and direct observation in confocal microscopy by anchoring them to traditional glass supports with a calibrated thickness of ∼50µm. The matrix composition can be readily adapted to specific neural cell types, notably by incorporating appropriate neurotrophic growth factors. Both PC-12 and SH-SY5Y lines respond to growth factors (nerve growth factor and brain-derived neurotrophic factor, respectively) impregnated and slowly released from the support. Significant neurite outgrowth is reported for a large proportion of cells, up to 66% for PC12 and 49% for SH-SY5Y. It is also shown that both growth factors can be chemically conjugated (EDC/NHS) throughout the matrix and yield similar proportions of cells with longer neurites (61% and 52%, respectively). Finally, neurite outgrowth was observed over several tens of microns within the 3D matrix, with both diffusing and immobilized growth factors.

Coagulation factor X mediates adenovirus type 5 liver gene transfer in non-human primates (Microcebus murinus).

Coagulation factor X (FX)-binding ablated adenovirus type 5 (Ad5) vectors have been genetically engineered to ablate the interaction with FX, resulting in substantially reduced hepatocyte transduction following intravenous administration in rodents. Here, we quantify viral genomes and gene transfer mediated by Ad5 and FX-binding-ablated Ad5 vectors in non-human primates. Ad5 vectors accumulated in and mediated gene transfer predominantly to the liver, whereas FX-binding-ablated vectors primarily targeted the spleen but showed negligible liver gene transfer. In addition, we show that Ad5 binding to hepatocytes may be due to the presence of heparan sulfate proteoglycans (HSPGs) on the cell membrane. Therefore, the Ad5-FX-HSPG pathway mediating liver gene transfer in rodents is also the mechanism underlying Ad5 hepatocyte transduction in Microcebus murinus.

The grey mouse lemur: a non-human primate model for ageing studies.

The use of non-human primate models is required to understand the ageing process and evaluate new therapies against age-associated pathologies. The present article summarizes all the contributions of the grey mouse lemur Microcebus murinus, a small nocturnal prosimian primate, to the understanding of the mechanisms of ageing. Results from studies of both healthy and pathological ageing research on the grey mouse lemur demonstrated that this animal is a unique model to study age-dependent changes in endocrine systems, biological rhythms, thermoregulation, sensorial, cerebral and cognitive functions.

[Anti-TNF alpha monoclonal antibodies (infliximab) and tuberculosis: apropos of 3 cases]. / Anticorps anti-TNF alpha (infliximab) et tuberculose: à propos de trois cas.

INTRODUCTION: Monoclonal TNF alpha antibodies are a new treatment of severe rheumatoid arthritis. One of the possible side effects is the appearance of opportunistic infections. We report here on three cases of disseminated tuberculosis observed in patients undergoing treatment with infliximab. EXEGESIS: A 45-year-old woman, treated with infliximab, was hospitalised after five infusions for fever and dyspnoea. The exams showed pulmonary and peritoneal tuberculosis. The second case is a 75-year-old woman whose symptoms were fever, cough and cervical adenopathy after three infliximab infusions. Diagnosis was disseminated tuberculosis. The third case is a 59-year-old man who was hospitalised for an infectious syndrome with dyspnoea, after two infliximab infusions. We discovered pulmonary tuberculosis. CONCLUSION: These three cases added to the 68 cases of tuberculosis registered with the treatment of infliximab. This confirms the risk of severe opportunist infectious side effects. TNF alpha is a cytokine which has anti-infectious properties. These tuberculoses are severe and generalized. It is recommended to search for an active or latent tuberculosis before beginning treatment with infliximab, and to check these patients frequently.

Three-dimensional structure of the lithostathine protofibril, a protein involved in Alzheimer's disease.

Neurodegenerative diseases are characterized by the presence of filamentous aggregates of proteins. We previously established that lithostathine is a protein overexpressed in the pre-clinical stages of Alzheimer's disease. Furthermore, it is present in the pathognomonic lesions associated with Alzheimer's disease. After self-proteolysis, the N-terminally truncated form of lithostathine leads to the formation of fibrillar aggregates. Here we observed using atomic force microscopy that these aggregates consisted of a network of protofibrils, each of which had a twisted appearance. Electron microscopy and image analysis showed that this twisted protofibril has a quadruple helical structure. Three-dimensional X-ray structural data and the results of biochemical experiments showed that when forming a protofibril, lithostathine was first assembled via lateral hydrophobic interactions into a tetramer. Each tetramer then linked up with another tetramer as the result of longitudinal electrostatic interactions. All these results were used to build a structural model for the lithostathine protofibril called the quadruple-helical filament (QHF-litho). In conclusion, lithostathine strongly resembles the prion protein in its dramatic proteolysis and amyloid proteins in its ability to form fibrils.

Lithostathine and pancreatitis-associated protein are involved in the very early stages of Alzheimer's disease.

According to one of the theories formulated to explain the etiology of Alzheimer's disease (AD), amylosis may reflect a specific inflammatory response. Two inflammatory proteins, lithostathine and PAP, were evidenced by immunohistochemistry in senile plaques and neurofibrillary tangles of patients with AD. In addition, lithostathine and PAP were significantly increased in the cerebrospinal fluid of patients with AD when compared to patients with multiple sclerosis, another inflammatory disease, and to normal control subjects. However, no correlation was observed with age of occurrence. Furthermore, lithostathine and PAP were increased even at the very early stages of AD, and their level remained elevated during the course of the AD unlike TNFalpha whose level, very high at very early stages, regularly decreased. Finally, if part of lithostathine and PAP are synthesized in the brain, a large part comes from serum by passage over the blood-brain barrier. These results indicate (i) the existence of an acute phase response followed by a chronic inflammation in AD, and (ii) that lithostathine and PAP are involved even at the first pre-clinical biochemical events of AD. In addition, because lithostathine undergoes an autolytic cleavage leading to its precipitation and the formation of fibrils, we believe that it may be involved in amyloidosis and tangles by allowing heterogeneous precipitation of other proteins.

[Long-term treatment of Alzheimer's disease: followup of a cohort of 255 patients treated with lacrine for four years]. / Traitement de la maladie d'Alzheimer au long cours: à propos d'une cohorte de 255 patients traités quatre ans par tacrine.

We describe the follow-up of a cohort of 255 Alzheimer's disease (AD) patients (81 males, 174 females) treated by tacrine during 4 years. We performed the survey of hepatic, cholinergic and general tolérance. Drug efficacy was measured by MMS examination on weeks 0, 18, 30, 52, 104, 156 and 208. A total of 190 patients (74.5 percent) were dropped out of this study, 75 (29 percent) for adverse events. We found 85 hepatic (33 percent), 79 cholinergic (31 percent), 31 (12 percent) neuropsychiatric and 72 general (28 percent) side effects. In term of drug efficacy we observed a global decline of 2.5 MMS points during the first year and 2 MMS points between W52 and W156. Tacrine's symptomatic efficacy, defined as the number of patients improved or stabilized at W30, was present in 50 patients (46 percent) among the 109 patients reaching W30. The intensity of symptomatic efficacy was expressed by a 2.7 MMS points increase in 37 patients improved on W30. The long term effects of Tacrine, measured by the MMS score at one year, showed a positive impact as the MMS was 2.5 points above the expected score in non treated AD patients. This study raises the practical problem of optimal cholinesterase inhibitors use in AD and the theoretical question of long term action of cholinesterase inhibitors on cerebral lesions of AD.

Association of APOE promoter but not A2M polymorphisms with risk of developing Alzheimer's disease.

The APOE4 allele is widely accepted as a major risk factor for late-onset Alzheimer's disease (AD). Recently, it has been reported that polymorphisms in the APOE promoter and in the alpha2-macroglobulin gene (A2M) are associated with AD. We have analyzed the distribution of APOE alleles, -219T/G APOE promoter polymorphism, and A2M/A2Mdel polymorphism in a large case-control study. Our results showed that APOE genotype was the only informative marker of AD risk contrary to -219T/G and A2M/A2Mdel polymorphism. In AD patients however, a strong linkage disequilibrium was observed between the T allele of -219T/G polymorphism and APOE4 allele. This result indicates that -219T/G APOE promoter polymorphism is a risk factor for AD by increasing the APOE4-associated risk.

Mechanism of calcite crystal growth inhibition by the N-terminal undecapeptide of lithostathine.

Pancreatic juice is supersaturated with calcium carbonate. Calcite crystals therefore may occur, obstruct pancreatic ducts, and finally cause a lithiasis. Human lithostathine, a protein synthesized by the pancreas, inhibits the growth of calcite crystals by inducing a habit modification: the rhombohedral (10 14) usual habit is transformed into a needle-like habit through the (11 0) crystal form. A similar observation was made with the N-terminal undecapeptide (pE(1)R(11)) of lithostathine. We therefore aimed at discovering how peptides inhibit calcium salt crystal growth. We solved the complete x-ray structure of lithostathine, including the flexible N-terminal domain, at 1.3 A. Docking studies of pE(1)R(11) with the (10 14) and (11 0) faces through molecular dynamics simulation resulted in three successive steps. First, the undecapeptide progressively unfolded as it approached the calcite surface. Second, mobile lateral chains of amino acids made hydrogen bonds with the calcite surface. Last, electrostatic bonds between calcium ions and peptide bonds stabilized and anchored pE(1)R(11) on the crystal surface. pE(1)R(11)-calcite interaction was stronger with the (11 0) face than with the (10 14) face, confirming earlier experimental observations. Energy contributions showed that the peptide backbone governed the binding more than did the lateral chains. The ability of peptides to inhibit crystal growth is therefore essentially based on backbone flexibility.

Biophysical characterization of lithostathine. Evidences for a polymeric structure at physiological pH and a proteolysis mechanism leading to the formation of fibrils.

Lithostathine is a calcium carbonate crystal habit modifier. It is found precipitated under the form of fibrils in chronic calcifying pancreatitis or Alzheimer's disease. In order to gain better insight into the nature and the formation of fibrils, we have expressed and purified recombinant lithostathine. Analytical ultracentrifugation and quasi-elastic light scattering techniques were used to demonstrate that lithostathine remains essentially monomeric at acidic pH while it aggregates at physiological pH. Analysis of these aggregates by electron microscopy showed an apparently unorganized structure of numerous monomers which tend to precipitate forming regular unbranched fibrils. Aggregated forms seem to occur prior to the apparition of fibrils. In addition, we have demonstrated that these fibrils resulted from a proteolysis mechanism due to a specific cleavage of the Arg(11)-Ile(12) peptide bond. It is deduced that the NH(2)-terminal undecapeptide of lithostathine normally impedes fiber formation but not aggregation. A theoretical model explaining the formation of amyloid plaques in neurodegenerative diseases or stones in lithiasis starting from lithostathine is described. Therefore we propose that lithostathine, whose major function is unknown, defines a new class of molecules which is activated by proteolysis and is not involved in cytoskeleton nor intermediate filament functions.

Nucleation of calcium oxalate crystals by albumin: involvement in the prevention of stone formation.

BACKGROUND: Urine is supersaturated in calcium oxalate, which means that it will contain calcium oxalate crystals that form spontaneously. Their size must be controlled to prevent retention in ducts and the eventual development of a lithiasis. This is achieved, in part, by specific inhibitors of crystal growth. We investigated whether promoters of crystal nucleation could also participate in that control, because for the same amount of salt that will precipitate from a supersaturated solution, increasing the number of crystals will decrease their average size and facilitate their elimination. METHODS: Albumin was purified from commercial sources and from the urine of healthy subjects or idiopathic calcium stone formers. Its aggregation properties were characterized by biophysical and biochemical techniques. Albumin was then either attached to several supports or left free in solution and incubated in a metastable solution of calcium oxalate. Kinetics of calcium oxalate crystallization were determined by turbidimetry. The nature and efficiency of nucleation were measured by examining the type and number of neoformed crystals. RESULTS: Albumin, one of the most abundant proteins in urine, was a powerful nucleator of calcium oxalate crystals in vitro, with the polymers being more active than monomers. In addition, nucleation by albumin apparently led exclusively to the formation of calcium oxalate dihydrate crystals, whereas calcium oxalate monohydrate crystals were formed in the absence of albumin. An analysis of calcium oxalate crystals in urine showed that the dihydrate form was present in healthy subjects and stone formers, whereas the monohydrate, which is thermodynamically more stable and constitutes the core of most calcium oxalate stones, was present in stone formers only. Finally, urinary albumin purified from healthy subjects contained significantly more polymers and was a stronger promoter of calcium oxalate nucleation than albumin from idiopathic calcium stone formers. CONCLUSIONS: Promotion by albumin of calcium oxalate crystallization with specific formation of the dihydrate form might be protective, because with rapid nucleation of small crystals, the saturation levels fall; thus, larger crystal formation and aggregation with subsequent stone formation may be prevented. We believe that albumin may be an important factor of urine stability.

An immune response to ice crystals in North Atlantic fishes.

In mammals, the presence of crystals composed of small organic molecules, including urate and related compounds, has been shown to trigger an inflammatory response and the subsequent production of specific immunoglobulins (Ig's). Many fishes that are exposed to ice crystals in cold temperate and polar oceans may harbour ice crystals internally. Here, we report evidence for a specific immune response to ice crystals in cold-ocean marine fishes. Using ice nucleation activity as an assay, anti-ice Ig's were detected in the sera of the cold-ocean marine fish species, ocean pout (Macrozoarces americanus) and Atlantic herring (Clupea harengus harengus), but not in the sera of species that are not exposed to ice. Purified Ig's isolated from ocean pout serum using two different protocols showed ice nucleation activity, thus demonstrating the presence of ice binding specificity among these Ig's.

Pancreatic lithostathine as a calcite habit modifier.

Most biological fluids are supersaturated with calcium salts. A mechanism controlling crystal growth is therefore necessary to prevent excessive precipitation and development of a lithiasis. In pancreatic juice, calcite precipitation is prevented by lithostathine, a glycoprotein that inhibits calcite crystal growth. We describe here the interaction of lithostathine with calcite crystals. Without lithostathine, calcite crystals grew as rhombohedra showing six (104) faces. At low concentration (1 microM), lithostathine already altered crystal growth by generating new (110) faces. At physiological concentrations (3-10 microM), adsorption resulted in a transition from rhombohedral to sub-cubic habits. Immunochemical localization demonstrated that, although all (104) faces are equivalent, lithostathine binding was restricted to the face edges distal to the c axis. Scanning electron microscopy showed that, at the site of lithostathine binding, spreading of new CaCO3 layers during crystal growth was arrested before reaching the crystal diad axis-bearing edges. The successive kinks generated during crystal growth formed the new, striated (110)faces. Similar modifications were observed with the N-terminal undecapeptide of lithostathine that bears the inhibitory activity. With 100 microM lithostathine, (110) faces could reach the c axis outcrop of the former rhombohedron, resulting in an olive-shaped crystal. Finally, the number of crystals increased and their average size decreased when lithostathine concentration increased from 0.1 to 100 microM. Decreased Ca2+ concentration during crystal growth was delayed in the presence of lithostathine. It was concluded that lithostathine controls lithogenesis 1) by triggering germination of numerous calcite crystals and 2) by inhibiting the rate of Ca2+ ion apposition on the nuclei and therefore interfering with the apposition of new layers on calcite. Formation of smaller crystals, whose elimination is easier, is thereby favored.

Calcium carbonate crystals promote calcium oxalate crystallization by heterogeneous or epitaxial nucleation: possible involvement in the control of urinary lithogenesis.

A large proportion of urinary stones have calcium oxalate (CaOx) as the major mineral phase. In these stones, CaOx is generally associated with minor amounts of other calcium salts. Several reports showing the presence of calcium carbonate (CaCO3) and calcium phosphate in renal stones suggested that crystals of those salts might be present in the early steps of stone formation. Such crystals might therefore promote CaOx crystallization from supersaturated urine by providing an appropriate substrate for heterogeneous nucleation. That possibility was investigated by seeding a metastable solution of 45Ca oxalate with vaterite or calcite crystallites. Accretion of CaOx was monitored by 45Ca incorporation. We showed that (1) seeds of vaterite (the hexagonal polymorph of CaCO3) and calcite (the rhomboedric form) could initiate calcium oxalate crystal growth; (2) in the presence of lithostathine, an inhibitor of CaCO3 crystal growth, such accretion was not observed. In addition, scanning electron microscopy demonstrated that growth occurred by epitaxy onto calcite seeds whereas no special orientation was observed onto vaterite. It was concluded that calcium carbonate crystals promote crystallization of calcium oxalate and that inhibitors controlling calcium carbonate crystal formation in Henle's loop might play an important role in the prevention of calcium oxalate stone formation.

Crystal structure of human lithostathine, the pancreatic inhibitor of stone formation.

Human lithostathine (HLIT) is a pancreatic glycoprotein which inhibits the growth and nucleation of calcium carbonate crystals. The crystal structure of the monomeric 17 kDa HLIT, determined to a resolution of 1.55 angstroms, was refined to a crystallographic R-factor of 18.6%. Structural comparison with the carbohydrate-recognition domains of rat mannose-binding protein and E-selectin indicates that the C-terminal domain of HLIT shares a common architecture with the C-type lectins. Nevertheless, HLIT does not bind carbohydrate nor does it contain the characteristic calcium-binding sites of the C-type lectins. In consequence, HLIT represents the first structurally characterized member of this superfamily which is not a lectin. Analysis of the charge distribution and calculation of its dipole moment reveal that HLIT is a strongly polarized molecule. Eight acidic residues which are separated by regular 6 angstrom spacings form a unique and continuous patch on the molecular surface. This arrangement coincides with the distribution of calcium ions on certain planes of the calcium carbonate crystal; the dipole moment of HLIT may play a role in orienting the protein on the crystal surface prior to the more specific interactions of the acidic residues.

Crystallization and preliminary crystallographic study of human lithostathine.

Crystals of human lithostathine, a pancreatic glycoprotein which inhibits the growth and nucleation of calcium carbonate crystals, were grown using PEG 4000 as the precipitating agent. The crystals belong to the hexagonal space group P6(1) (or its enantiomorph P6(5)) and diffract to 1.55 A resolution. There is one molecule in the asymmetric unit and the crystals have 39% solvent.

Analysis of the soluble organic matrix of five morphologically different kidney stones. Evidence for a specific role of albumin in the constitution of the stone protein matrix.

Our aims were to analyze the protein composition of the organic matrix of urinary stones and to investigate the role of albumin in its constitution. Five different morphological types of stones were studied. Proteins extracted from the stone were submitted to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by immunoblotting with antibodies to 13 urinary proteins. Nine of the 13 proteins were found in all types of stone: human serum albumin (HSA), alpha 1-acid glycoprotein (alpha 1-GP), alpha 1-microglobulin (alpha 1-M), immunoglobulins (Igs), apolipoprotein A1 (apo-A1), transferrin (Tr), alpha 1-antitrypsin (alpha 1-T), retinol-binding protein (RBP) and renal lithostathine (RL). The beta 2-microglobulin (beta 2-M) was present only in calcium oxalate and uric acid stones. In contrast, ceruloplasmin, haptoglobin and Tamm-Horsfall protein (THP) were detected in none of them. Because HSA appeared as the major protein component in all stones, we wondered whether it might play a specific role in the constitution of the stone matrix. Association of HSA with urinary proteins that were present in stones was demonstrated by showing that proteins present in the matrix comigrated with HSA on gel filtration, whereas proteins that were absent did not. Moreover, HSA induced the binding of stone matrix proteins to an albumin-specific affinity column. Finally, we evidenced HSA binding to calcium oxalate monohydrate (COM) crystals in a solution similar to urine.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein inhibitors of calcium salt crystal growth in saliva, bile and pancreatic juice.

The control of the formation of crystals in biological fluids is one of the most exciting field of research involving both organic and biochemical areas. Many organisms have evolved mechanisms which minimize or avoid the effects of nucleation and crystal growth formation. One of the most important mechanism is the interaction of specific proteins, called inhibitors, with crystals which alters their habits and leads to their elimination. This article, focused on saliva, pancreatic juice and bile, reviews our present knowledge on the structure-function relationships existing between these proteins and their ability to inhibit the growth of different calcium salt crystals.