[Evolutionary comparison of laboratory cardiac and infectious parameters during septic shock: about an original case]. / Evolution comparée des indicateurs biologiques cardiaques et infectieux lors du choc septique: à propos d'une observation originale.

A massive release of troponin Ic and CKMB was described in a patient during septic shock. According to experimental animal models previously described, this release of biological markers by myocardial tissue could be due to an inflammatory process of myocardial tissue during septic shock without myocardial infarction in non cardiac critically ill patients.

[About two cases of massive hypercholesterolemia]. / A propos de deux cas d'hypercholestérolémie massive.

Two patients presented massive hypercholesterolemia related to acquired disease, intrahepatic cholangitis with cholestasis in one and nephrotic syndrome in the second. Comparison of the lipoprotein patterns demonstrated distinctive pathophysiological processes different from those operating in primary causes of hypercholesterolemia.

[Unexpected occurence of hemoglobin Athens-Georgia]. / Révélation inattendue de l'hémoglobine Athens-Georgia.

The following report concerned a 47 year old Caucasian diabetic patient. Routine HPLC of HbA1c (Variant II Biorad Laboratories - hemoglobin A1c program) resulted only in the evidence of HbF (1%) and increase in HbA1c (10%). Considering the presence of HbF a standard agarose gel electrophoresis of patient's hemoglobin was performed and revealed the presence of Hb Athens-Georgia. Consequently the occurrence of HbF during determination of HbA1c by HPLC should lead to perform a standard hemoglobin electrophoresis in order to explore an hidden, unsuspected and clinically silent occurrence of rare Hb variant or additional unsuspected increase in HbA2.

Physiological strains induce differentiation in human osteoblasts cultured on orthopaedic biomaterial.

We have developed an in vitro mechanical stretching model of osteoblastic cells cultured on metallic biomaterials in order to study the effects of mechanical strain on osteointegration of orthopaedic implants. Titanium alloy discs coated with alumina or hydroxyapatite were used as substrates. Three Dynacell devices were especially designed to apply cyclic strains on rigid biomaterials. The regimen (600 mu epsilon strains, 0.25Hz) was defined on the basis of physiological data and estimated deformation on hip stem prostheses. The performances of these apparatus were reproducible and provided controlled deformations. Human osteosarcoma cell line MG-63, human osteoblasts obtained from primary cultures and ROS 17/2.8 rat osteosarcoma cells were used as cell models. Cell behaviour was assessed in terms of growth and alkaline phosphatase (ALP) activity by in situ assays for two regimens: 15-min deformations repeated three times a day to mimic rehabilitation exercises and 24-h continuous deformations. We demonstrated that continuous deformation did not affect the growth and ALP activity of MG-63 cells, in contrast with sequential deformations which had no effect on cell number, but which stimulated ALP activity after 5 days of stretching. This sequential regimen can also modify the behaviour of human bone-derived cells resulting in increased proliferation after 5 days and stimulation of ALP activity after 15 days. ROS 17/2.8 rat osteosarcoma cells submitted to sequential deformations responded faster than other cell lines by increasing their ALP activity only after 1 day of stretching. Like MG-63 cells, proliferation of the ROS 17/2.8 rat osteosarcoma cell line was not affected by sequential deformations. This study suggests that short, repeated deformations defined to mimic rehabilitation exercises recommended after prostheses implantation are more likely to exert beneficial effects on implanted bone than continuous strains.

In vitro glycoxidation of insoluble fibrous type I collagen: solubilization and advanced glycation end products.

The deleterious effects of glycoxidation are dependent on the half-life of proteins. Collagen, the main component of extracellular matrices, is a long live protein and thus may be sensitive to the glycoxidation process. We incubated calf skin fibrous type I collagen in PBS at 37 degrees C with glucose. The fibrous type I collagen was solubilized and an increase in the amount of advanced glycation end products of the solubilized fraction was observed. As there was no bacterial contamination and no proteolytic activities in the incubation medium, the solubilization of fibrous type I collagen is probably due to the speculative production of the free radicals in our experimental conditions. To test this hypothesis, fibrous type I collagen was incubated in PBS with AAPH (2,2'azo-bis 2-aminodinopropane) a free radicals generator. AAPH induced a dramatic and dose dependent solubilization of fibrous type I collagen.

Serum pentosidine as an indicator of Alzheimer's disease.

Pentosidine, an advanced glycation end product (AGE), was assayed by HPLC in serum proteins from patients with Alzheimer type dementia (AD), patients with diabetes mellitus (D), and healthy (C) age-matched old subjects (mean age from each group = 84 years). Serum pentosidine was significantly different between the three groups despite similar renal function (serum creatinine < 160 micromol/L). In all groups of patients, pentosidine was independent of glycated hemoglobin (HbA1C) and the early glycation marker fructosamine and appeared to be an independent marker, mainly bound to serum albumin. Pentosidine could be an important factor useful for the diagnosis of Alzheimer's disease.

Nutritional markers, acute phase reactants and tissue inhibitor of matrix metalloproteinase 1 in elderly patients with pressure sores.

BACKGROUND: Loss of collagen and elastin is observed in the elderly. In geriatric inpatients, chronic protein malnutrition could induce susceptibility to additional morbidity such as pressure sores. OBJECTIVE: The purpose of this study was to explore the relationship between nutritional and inflammatory status and the production of tissue inhibitor of matrix metalloproteinase 1 (TIMP-1). METHODS: Chronically ill elderly inpatients, without or with pressure sores, were enrolled. Nutritional protein markers, acute phase reactants, and TIMP-1 were determined, and changes in these biological parameters were compared. RESULTS: Chronic inflammatory process and protein malnutrition were observed in all enrolled patients. The severity of these two pathophysiological processes was independent of the occurrence of pressure sores. The serum prealbumin and albumin levels were lower in patients with pressure sores than in those without. In addition, the general increase in the TIMP-1 level was independent of the occurrence of pressure sores. The TIMP-1 level was mainly related to the prognostic inflammatory and nutritional index. CONCLUSIONS: The general increase in acute-phase reactants observed in the elderly could be related to the intercurrent diseases. The generally low serum albumin level, lowest in patients with pressure sores, may be considered evidence of protein malnutrition and hypercatabolism. Regarding the morbidity, the increase in TIMP-1 levels could be explained as an adaptive process to prevent intrinsic protein expenditure of extracellular matrices.

Production of matrix metalloproteinase 2 in fibroblast reaction to mechanical stress in a collagen gel.

The reaction of fibroblasts to mechanical forces generated by fibroblasts themselves in anchored collagen lattices was studied. Fibroblasts were cast in collagen gels. Free retracted gels (RG) were compared with stressed gels (SG) in 3-day and 14-day experiments. As previously described, SG showed an increase in protein, mainly collagen, biosynthesis. Matrix metalloproteinases (pro-MMP-2 and MMP-2) were studied by zymography. Certain cell membrane components, integrin alpha(2) and phosphatidylserine, were studied by flow cytometry with antibodies against the integrin alpha(2) subunit and with annexin V binding. Mechanical stress stimulated production of pro-MMP-2 both in the short-term (3-day) and the longer term (14-day) cultures. However, the pro-enzyme was not more activated and there was no difference in the amount of MMP-2 between RG and SG. There was only an increase with time under both conditions. The stressed fibroblasts reacted early with an increase in the integrin alpha(2) subunit, but the stimulated cells disappeared from the 14-day cultures. The number of cells measured in terms of the amount of DNA decreased between day 3 and day 14, mainly in the SG due to cytolysis. This cell stress was related to an alteration in the plasma membrane detected by the annexin V marker.

Synergy between ascorbate and alpha-tocopherol on fibroblasts in culture.

Ascorbate and tocopherol are important antioxidants that protect cells against oxidative stress. The interaction of ascorbate and alpha-tocopherol in cells is difficult to detect as both ascorbate and alpha-tocopherol are unstable in vitro in a biological medium. We examined the interactions between human dermal fibroblasts, ascorbate and alpha-tocopherol to determine the effects of the vitamins on growth and cell viability. The interaction of ascorbate and alpha-tocopherol was studied in a fibroblast culture medium during 48h. Ascorbate and alpha-tocopherol were detected by fluorimetry after high-performance liquid chromatography (HPLC). Cell growth and cell viability were studied by cell numeration after trypan blue staining. The ascorbate concentration fell in presence of alpha-tocopherol in cell culture medium under all experimental conditions, with or without cells. Ascorbate partly protected alpha-tocopherol but only in presence of cells. Cell viability was preserved by alpha-tocopherol whereas ascorbate enhanced fibroblast growth. The synergy between ascorbate and alpha-tocopherol corresponds to a consumption of ascorbate which spares alpha-tocopherol but only in presence of cells.

Monitoring of ascorbate at a constant rate in cell culture: effect on cell growth.

Ascorbic acid (vitamin C) is a primary antioxidant for cells. But, ascorbic acid added to culture medium is not readily available to cells in culture, because it is unstable in aqueous media. We determined the conditions required to obtain and maintain a constant concentration of ascorbate in the culture medium using ascorbate and ascorbate-phosphate. The study was carried out with human fibroblasts and the amounts of ascorbate in the culture medium were determined by high performance liquid chromatography. A mixture of 0.25 mmol/L ascorbate and 0.45 mmol/L ascorbate-phosphate provided a constant concentration of ascorbate in the culture medium. This constant ascorbate concentration proved to be nontoxic for cells and stimulated cell growth in the short term and long term.

Practice of a testing bench to study the effects of cyclic stretching on osteoblast-orthopaedic ceramic interactions.

A new experimental method has been used to study the behaviour of human osteoblasts cultured on bioceramics subjected to mechanical strains. The ceramics were alumina, hydroxyapatite (HA) and a duplex system composed of hydroxyapatite-covered alumina. The system applied 400 microdeformations for a 6-h period with a cycle frequency of 0.5 Hz to osteoblasts growing on ceramic-covered disks. The effects of strains on short-term cell viability, cell growth, alkaline phosphatase (ALP) activity, and collagen biosynthesis were assessed. When possible, the parameters (lactate dehydrogenase) were studied along the experiment in samples of the culture medium, in the other cases by comparison of stretched and unstretched cultures on the same ceramics with the same cell line. In relationship with the coating, mechanical strains resulted in a decrease in DNA corresponding to cell number, an LDH release during straining, an unchanged (alumina) or decreased (HA and duplex) ALP activity, a decrease (HA and duplex) of collagen and total protein synthesis or an increase of it (alumina). The stress-producing device and its associated protocol are shown to be suitable for investigating the behaviour of cells, cultured on biomaterials subjected to mechanical strain.

Mineralization and alkaline phosphatase activity in collagen lattices populated by human osteoblasts.

Adult human osteoblastic cells were grown in a native type I collagen gel. Proliferation and viability analyses showed that cells rapidly stopped dividing and became blocked in the G0G1 phase (91% on day 13). Carboxyfluorescein diacetate cell staining and flow cytometry showed that osteoblasts were viable for the first 16 days and then viability decreased (58% viable cells on day 22). Osteoblasts were able to retract the matrix. Betaglycerophosphate (betaGP) stimulated the deposition of mineral particles in the collagen network, and electron probe microanalysis showed that they were principally calcium and phosphorus, with a Ca/P ratio of about 1.7. Various times of betaGP supply were tested. We compared 10 mM betaGP added only once at day 0, or continuously from day 0, day 8, or day 21. Mineralization was observed in conditions where betaGP was added at day 0. Furthermore, 10 mM betaGP added once during gel preparation was sufficient to induce mineralization with mineral accumulation up to day 15 whereas the speed of the gel contraction decreased. In every condition, cultures expressed high alkaline phosphatase (ALP) levels as early as day 3, which decreased afterwards. These kinetics might explain why the other conditions did not prove favorable to the mineralization process. The model was used to study the influence of blocking gel retraction. Blocking retraction delayed the ALP activity decrease, but had no effect on mineralization. In conclusion, human adult osteoblasts cultured in native collagen gel stopped proliferation and underwent mineralization very early. This model should be used to investigate the influence of effectors on the early stages of culture.

Urine glycyl-L-proline increase and skin trophicity.

Glycyl-L-proline (gly-pro) is an end product of collagen metabolism that is further cleaved by prolidase (EC 3.4.13.9); the resulting proline molecules are recycled into collagen or other proteins. We postulated a relationship between defective gly-pro hydrolysis, increased collagen degradation and skin destruction. This relationship was tested using HPLC to measure the gly-pro in urine. 24 hour urine samples were collected from 27 old people (86 +/- 6 years old), of whom 15 were suffering from skin pressure sores of the sacrum or calcaneus. The urine from patients with pressure sores contained significantly more gly-pro than the urine from the control. A cut-off at 7 mumol/mmol creatinine gave the test a positive predictive value of 70%. Collagen breakdown was also increased as indicated by the increase of hydroxyproline (hyp) in the urine. But this breakdown seemed to stop at the gly-pro step.

Quantitative zymography of matrix metalloproteinases by measuring hydroxyproline: application to gelatinases A and B.

Gelatinases A and B are metalloproteinases involved in the degradation of the extracellular matrix. Detection and quantification of these enzymes in physiological and pathological conditions such as rheumatoid arthritis, tumor invasion and metastasis may be clinically useful. Gelatin zymography is an electrophoretic technique specific for gelatinases. It can be used to detect the activity of both the active and latent forms. We have standardized this technique for the active and latent forms of gelatinase A and for the latent form of gelatinase B. We measured the extent of gelatin degradation with an EDC scanning densitometer (Helena). The value recorded was directly proportional to the amount of enzyme. Gelatinase activity was quantified from the gel by assaying hydroxyproline as an index of gelatin breakdown. Gelatin zymography was found to be useful in characterizing gelatinases A and B by their molecular weights and measuring their specific activity by a standardized analysis of the degraded gelatin substrate.

Interaction of a plasma-sprayed hydroxyapatite coating in contact with human osteoblasts and culture medium.

The loss of calcium from plasma-sprayed calcium phosphate ceramics (CPCs) on bioinert metal substrate (Ti-6Al-4V) immersed in cell culture medium with or without human osteoblast culture was measured. The ceramics were a CPC and a duplex system composed of a CPC layer on an alumina coating. The dissolution of calcium compounds was monitored by measuring the calcium leaked from the coatings into the culture medium in 15 days. Calcium was measured by flame photometry. The surfaces of the ceramics exposed to the culture medium and in contact with osteoblasts were analysed by X-ray diffraction (XRD). The dissolution process occurred in the first 6 days of contact, but the calcium released into the culture medium was only a small fraction of the calcium content of the coatings. The presence or absence of osteoblasts on the surface of the ceramics did not make significant difference for the calcium release. The XRD spectra of the ceramics before and after immersion and in contact with cells did not show a significant change in the compounds of the coatings.

A comparison of 15N proline and 13C leucine for monitoring protein biosynthesis in the skin.

The tracers L 15N-proline and L(1-13C)-leucine were used to explore the synthesis of skin proteins in vivo in rabbits. They orally received a single dose containing an equimolecular mixture of L(1-13C)-leucine and L 15N-proline. The changes in the amounts of these tracers in blood and skin were monitored for a total of 8 h. The data showed the appearance of the two tracers in blood within 15 min and their clearance in 8 h. They were both rapidly (15 min) incorporated into skin proteins, but more proline was incorporated than leucine. We therefore consider L 15N-proline to be a better tracer than L(1-13C)-leucine for studying protein metabolism in the skin.

Physiopathogenic investigations in a case of familial stiff-skin syndrome.

BACKGROUND: Stiff-skin syndrome (SSS) is a rare cutaneous syndrome characterized by stony-hard skin and limitation of joint mobility. Its cause is still unknown. OBJECTIVE: Biological investigations were performed in a new case of SSS. METHODS: Collagen production and DNA biosynthesis were studied from fibroblast culture. Proinflammatory cytokines (TNF-alpha, IL-6 and TGF-beta2) were measured in the patient's serum. Results were compared with pathological findings. RESULTS: Collagen production and DNA biosynthesis were normal whereas the level of circulating cytokines was high. Histological examination of the skin showed mild fibrosis in the dermis whereas the fascia was not thickened. CONCLUSION: Our clinical and biological findings suggest that in this case, cutaneous changes may be related to an inflammatory process rather than to a primary fibroblast defect or a fascial abnormality as previously hypothesized.