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.

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.

Carcinogen-induced mutation spectrum in wild-type, uvrA and umuC strains of Escherichia coli. Strain specificity and mutation-prone sequences.

Forward mutations induced by the ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF) in the tetracycline resistance gene carried on plasmid pBR322 are shown to be dependent upon the induction of the host SOS functions in wild-type and umuC Escherichia coli cells. The mutation frequency in the umuC strain is equal to about 40% of the mutation frequency observed in the umu+ background. In the excision-repair-deficient uvrA mutant strain the mutagenic response is the same as in SOS-induced wild-type cells whether or not the uvrA bacteria are SOS-induced. Equal mutation frequencies are obtained in both the wild-type and the uvrA strains for equal modification levels although the survival of AAF-modified plasmid DNA is greatly reduced in the uvrA strain as compared to the wild-type strain. Sequence analysis of the mutations reveals that more than 90% of the N-Aco-AAF-induced mutations are frameshift mutations. Two types of mutational hotspots are observed occurring either at repetitive sequences or at non-repetitive sequences. Both types of mutants appear at similar locations and frequencies in both the wild-type and the uvrA strains. On the other hand, only the non-repetitive sequence mutants are obtained in the umuC background. These non-repetitive sequence mutants preferentially occur within the sequence 5' G-G-C-G-C-C 3' (the NarI restriction enzyme recognition sequence). The analysis of the -AAF binding spectrum to the same DNA fragment shows that there is no direct correlation between the modification spectrum and the mutation spectrum. We suggest that certain sequences are "mutation-prone" in the sense that only these sequences can be efficiently mutated as the result of an active processing mediated by specific proteins. When a sequence is said to be mutation-prone it probably corresponds to a particular structure that is induced within this sequence as a result of the binding to the DNA of the mutagen. This sequence-specific conformational change is the substrate for the protein(s) that fixes the mutation. The mutagenic processing pathway(s) is part of the cellular response to DNA-damaging agents (the so-called SOS response). Two pathways for frameshift mutagenesis are suggested by the data: an umuC-dependent pathway, which is involved in the mutagenic processing of lesions within repetitive sequences; an umuC-independent pathway responsible for the fixation of mutations within specific non-repetitive sequences.

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.

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.

[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.

[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.

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.

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.

[Macromolecular inhibitors of crystallization in saliva and bile]. / Les inhibiteurs macromoléculaires de cristallisation dans la salive et dans la bile.

The current knowledges concerning macromolecular inhibitors of crystallization in saliva and in bile are reviewed. In saliva, four families of inhibiting proteins have been evidenced: the statherins, the acidic proline-rich proteins, the cystatins and the histatins. These proteins inhibit the nucleation and the growth of calcium phosphate salts. In the bile, two families of proteins that inhibit the nucleation of calcium carbonate and cholesterol are present: the apolipoproteins and the calcium binding protein also called anionic polypeptide fraction. The structure-function relationships of these molecules are particularly stressed.

[On the ultrastructure of the cysticercoid of Hymenolepis stylosa (Cestoda: Cyclophyllidea) (author's transl)]. / Etude ultrastructurale du cysticercoïde de Hymenolepis stylosa (Cestoda, Cyclophyllidea).

Electron microscopy of the tegument of H. stylosa cysticercoid as compared with that of H. diminuta reveals great variations in the fine structure of the larval tegument of these two species. On the scolex and the inner cyst wall, the tegument of H. stylosa cysticercoid bears typical microtriches; at the level of the anterior pore, the microtriches are flexuous. The tegument of the outer cyst wall appears as a syncytial band with dense material which accumulates in the region below the unit membrane, and the tegument of the cercomer is increased by branchied microvillies. While, the scolex of H. diminuta cysticercoid is unarmed and the tegument of the outer cyst wall bears branched microvillies. The data permit the conclusion that the differentiation of tegumental structures is graduated from the scolex to the cercomer. This differenciation is more or less important according to the parasite and results of successive inductions which affect parts of the cysticercoid. The significance of these findings is discussed in terms of the possible function of these structures and the penetration of substances.

[Life cycle of Maupasina weissi Seurat, 1913, Subuluroidea Nematode, parasite of the elephant shrew (author's transl)]. / Cycle biologique de Maupasina weissi Seurat, 1913 (Nématode Subuluroidea), Ontogenèse des structures céphaliques.

The life cycle of Maupasina weissi Seurat, 1913, the parasite of the elephant shrew, has been experimentally obtained from the intermediate host Locusta migratoria. The biology of this Nematoda is considered as being more primitive than the Subuluridae: -- egg maturation in external environment is in fact necessary to the Maupasina larvae to penetrate into the insect, -- The different localizations of the infective larvae, such as mesenteron regeneration crypta, fat body, demonstrate that the parasite is not completely adaptated to its intermediate host, -- the ontogenesis of cephalic structures is characterized by an hypertrophy of the archaic structures mainly from cuticular origin.

Cloning of CDC33: a gene essential for growth and sporulation which does not interfere with cAMP production in Saccharomyces cerevisiae.

The CDC33 gene of Saccharomyces cerevisiae belongs to the class II 'START' genes. Its product is required for the initiation of a new cell division cycle (Hartwell, 1974). Many results suggest that the cAMP signalling pathway is one of the major controlling elements of 'START'. Components of this pathway are encoded by class II 'START' genes. The aim of the present study is to determine whether or not the CDC33 gene interferes with the cAMP signalling pathway. We report here the molecular cloning of the CDC33 gene by complementation of the cdc33-1 thermosensitive mutant. The identity of the cloned gene is confirmed by site-specific reintegration and segregation analysis. This gene is transcribed into a 900-nucleotides mRNA and appears to be relatively abundant in the cell. We also show that the CDC33 gene product is essential for sporulation. cdc33-1 mutant cells are able to enter into the resting state. The cAMP intracellular pool is not modified when the cdc33-1 mutant is shifted to the restrictive temperature. The cdc33-1 mutation is not suppressed by other known elements of the cAMP cascade. All these results suggest that the CDC33 'START' gene does not interfere with the cAMP signalling pathway which controls cell division.

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.

Rat pancreatic stone protein messenger RNA. Abundant expression in mature exocrine cells, regulation by food content, and sequence identity with the endocrine reg transcript.

We used a cDNA encoding the human pancreatic stone protein (PSP-S), the secretory inhibitor of CaCO3 crystal growth, as a probe for cloning rat PSP-S messenger RNA. Overlapping clones gave a mRNA sequence of 783 nucleotides encoding a preprotein of 165 amino acids including a prepeptide of 21 amino acids. Rat and human PSP-S showed 70% identity, and the mature proteins had the same length. PSP-S mRNA concentration was measured in the pancreas of rats adapted to diets containing 15, 25, or 70% protein. Compared with the 15% protein diet, concentration increased 3 and 12 times with the diets with 25 and 70% protein, respectively, which is 3 times higher than for serine proteases. A complete sequence identity was observed between the rat PSP-S transcript and the reg mRNA described by Terazono et al. (Terazono, K., Yamamoto, H., Takasawa, S., Shiga, K., Yonemura, Y., Tochino, Y., and Okamoto, H. (1988) J. Biol. Chem. 263, 2111-2114), which is expressed in regenerating pancreatic islets but not in mature islets. A specific role of the reg protein in islet regeneration was suggested. We found that PSP-S (reg) mRNA concentration was indeed increased in isolated regenerating islets. Yet, a transient increase was also observed in exocrine tissue during the initial phase of regeneration following pancreatectomy or acute pancreatitis, suggesting increased expression during cell dedifferentiation. It is concluded that, in mature pancreas, expression of the reg/PSP-S gene occurs primarily in acinar cells. The gene product, which encodes a secretory protein inhibiting CaCO3 crystal growth in juice, is unlikely to play a specific role in islet regeneration.

Preliminary treatment of urinary proteins improves electrophoretic analysis and immunodetection.

Analysis of urinary protein composition is an important tool in studies on renal physiology and physiopathology. Urine is, however, a complex mixture containing, besides protein, a variety of compounds such as salts, peptides, oligosaccharides, and glycosaminoglycans. Some of these compounds interfere with the electrophoretic migration of protein in sodium dodecyl sulfate-polyacrylamide gels and prevent correct analysis of the protein pattern. We describe a simple method for extracting urinary proteins that considerably improves their electrophoretic migration and subsequent immunodetection. This treatment involves ammonium sulfate fractionations (for precipitating proteins), EDTA (for inhibiting protein aggregation), and HCl hydrolysis (for removing glycosylaminoglycans). Recovery during extraction was found to be almost quantitative for total protein and three representative proteins: albumin, alpha 1-glycoprotein acid, and beta 2-microglobulin.

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.

[Beta-2-microglobulin and rheumatoid factor levels in the synovial fluid (author's transl)]. / Bêta-2-microglobuline et facteur rhumatoïde dans le liquide synovial. 85 dosages.

The object of this study was to determine whether levels of beta-2-microglobulin and of rheumatoid factor measured by an enzyme-immunoassay allowed good discrimination between inflammatory and degenerative arthropathies. A multiparametric study of synovial fluid was performed on 85 specimens from patients with rheumatoid arthritis, chondrocalcinosis, mechanical arthritis and traumatic arthopathies. A beta-2-microglobulin level of less than 4 mg/l is a result very mich in favor of a non-inflammatory arthropathy (48/49 cases). The quantification of intra-articular rheumatoid factor allows for reclassification in the context of sero-negative rheumatoid arthritis.