Subchondral bone alterations in a novel model of intermediate post traumatic osteoarthritis in mice.

In Post Traumatic Osteoarthritis (PTOA), hypomineralization and increased remodeling of the Subchondral bone (SB) are the first stages of tissue alterations. Although these alterations are well depicted and one of the main targets in OA intervention, the link between SB compositional and mechanical properties alterations during OA progression remains scarce in the literature. Here, we hypothesized that SB shows - right after the first sign of gait pattern changes - a decrease in SB tissue formation depicted by (i) a decrease in thickness, (ii) a lower nanoscopic stiffness, and (iii) a decrease in mineral and collagen maturity. To test our hypothesis, we investigated PTOA in female C57Bl6 mice's right knee (n = 13 control group [CL] and n = 27 PTOA group) by using Gait Analysis, Histomorphometry, Nanoindentation, and Raman Spectroscopy (RS). We showed (i) an increased OA histological grade, (ii) a decrease in Cartilage and SB thickness, and (ii) an increase of stance time and stride length on both limbs. The lateral condyle - where the main forces were applied - of mice with PTOA decreased in the degree of mineralization and crystal size and presented a lower Modulus of Elasticity (E). However, while no difference was observed regarding collagen or mineral-related compositional RS properties, we depicted higher crystallinity in the medial condyle than the lateral condyle in the PTOA group, which we did not observe in the control group. Our study depicts an early onset of intermediate PTOA where SB nanoscopic stiffness decreases while the degree of mineralization is not severely altered yet.

Emergency ward ultrasound: clinical audit on disinfection practices during routine and sterile examinations.

CONTEXT: In the emergency ward, where the use of ultrasound is common (including for sterile procedures), ward equipment is constantly exposed to high risks of microbiological contamination. There are no clear guidelines for disinfection control practices in emergency departments, and it is not known how emergency ward doctors follow good hygiene practices. METHOD: A multi-centre audit was conducted in 16 emergency services from Northern France regional hospitals, in form of a questionnaire. It was proposed to all emergency ward physicians. We excluded questionnaires when physicians mentioned that they did not use ultrasound on a daily basis. The questionnaire was designed using existing hygiene and ultrasound disinfection practices guidelines from varying French medical societies. It included three different clinical scenarios: (a) ultrasound on healthy skin, (b) on injured skin, and (c) ultrasound-guided punctures. All questions were closed-ended, with only one answer corresponding to the guidelines. We then calculated compliance rates for each question, each clinical situation, and an overall compliance rate for all the questions. RESULTS: 104 questionnaires were collected, and 19 were excluded. For the 85 analysed questionnaires, the compliance rates were 60.4% 95% CI [56.4-64.7] for ultrasound on healthy skin, 70.9% 95% CI [66.3-76.1] on injured skin and 69.4% 95% CI [65.1-73.6] for ultrasound-guided punctures. The overall compliance rate for the compliance questions was 66.1% 95% CI [62.8-69.1]. Analysis of the questionnaires revealed severe asepsis errors, misuse of gel, ignorance of infection control practices to be applied in the context of ultrasound-guided puncture and exposure of the probe to body fluids. CONCLUSION: This study details areas for quality improvement in the disinfection of emergency ultrasound scanner use. Consequently, we propose a standardized protocol based upon the recommendations used for the questionnaire drafting, with a visual focus on the low compliance points that have been revealed in this audit. This protocol has been distributed to all the medical emergency services audited and included in the emergency resident's ultrasound learning program.

Clinical and laboratory correlates of lung disease and cancer in adults with idiopathic hypogammaglobulinaemia.

Idiopathic hypogammaglobulinaemia, including common variable immune deficiency (CVID), has a heterogeneous clinical phenotype. This study used data from the national UK Primary Immune Deficiency (UKPID) registry to examine factors associated with adverse outcomes, particularly lung damage and malignancy. A total of 801 adults labelled with idiopathic hypogammaglobulinaemia and CVID aged 18-96 years from 10 UK cities were recruited using the UKPID registry database. Clinical and laboratory data (leucocyte numbers and serum immunoglobulin concentrations) were collated and analysed using uni- and multivariate statistics. Low serum immunoglobulin (Ig)G pre-immunoglobulin replacement therapy was the key factor associated with lower respiratory tract infections (LRTI) and history of LRTI was the main factor associated with bronchiectasis. History of overt LRTI was also associated with a significantly shorter delay in diagnosis and commencing immunoglobulin replacement therapy [5 (range 1-13 years) versus 9 (range 2-24) years]. Patients with bronchiectasis started immunoglobulin replacement therapy significantly later than those without this complication [7 (range 2-22) years versus 5 (range 1-13) years]. Patients with a history of LRTI had higher serum IgG concentrations on therapy and were twice as likely to be on prophylactic antibiotics. Ensuring prompt commencement of immunoglobulin therapy in patients with idiopathic hypogammaglobulinaemia is likely to help prevent LRTI and subsequent bronchiectasis. Cancer was the only factor associated with mortality. Overt cancer, both haematological and non-haematological, was associated with significantly lower absolute CD8(+) T cell but not natural killer (NK) cell numbers, raising the question as to what extent immune senescence, particularly of CD8(+) T cells, might contribute to the increased risk of cancers as individuals age.

Accuracy assessment of online glucose monitoring by a subcutaneous enzymatic glucose sensor during exercise in patients with type 1 diabetes treated by continuous subcutaneous insulin infusion.

AIM: Online continuous glucose monitoring (CGM) during physical exercise would be highly useful in patients with insulin-treated diabetes. For this reason, this study assessed whether such a goal could be reached with a subcutaneous 'needle-type' enzymatic sensor. METHODS: Ten patients (five women/five men), aged 51 ± 12 years, with type 1 diabetes for 24 ± 11 years treated by continuous subcutaneous insulin infusion (CSII) for more than 1 year (HbA1c: 7.5 ± 0.8%) performed a 30-min bout of exercise at a constant high-intensity load (15% above their individual ventilatory threshold) on a cycle ergometer. All patients wore a subcutaneous 'needle-type' enzymatic glucose sensor linked to a portable monitor (Guardian(®) RT, Medtronic-MiniMed, Northridge, CA, USA) that had been inserted the previous evening. Sensor calibration was performed against capillary blood glucose immediately before the exercise. CGM values were recorded every 5 min from T(-10) to T(+30), then every 10 min during the recovery period from T(+30) to T(+90). These recorded values were compared with blood glucose assays performed on simultaneously collected venous samples. RESULTS: Sensor functioning and tolerability raised no problems except for one sensor that could not be adequately calibrated. Data from this patient were excluded from the data analysis. An average blood glucose decrease of 63 ± 63 mg/dL (3.5 ± 3.5 mmol/L) (median decrease: 58 mg/dL [3.22 mmol/L]; range: -3 mg/dL [0.16 mmol/L] to 178 mg/dL [9.8 mmol/L]) occurred during exercise bouts, while CGM values decreased by 38 ± 49 mg/dL (2.11 ± 2.72 mmol/L) (median: 32 mg/dL [1.7 mmmol/L]; range: -15 mg/dL [0.83 mmol/L] to 58 mg/dL [3.22 mmol/L]). Cumulative paired glucose values (n = 135) could be analyzed. The correlation factor between CGM and blood glucose values was 0.957 with an intercept of 0.275. The mean difference between paired values according to Bland-Altman analysis was 10 ± 31 mg/dL (0.56 ± 1.72 mmol/L). Clarke error grid analysis showed 91% of paired points in A and B zones, while 0%, 9% and 0% of paired points were in the C, D and E zones, respectively. CONCLUSION: Blood glucose changes during intensive physical-exercise bouts performed by CSII-treated type 1 diabetes patients can be estimated with acceptable clinical accuracy by online CGM.

Actinides and decay products in selected produce and bioindicators in the vicinity of a uranium plant.

Cypress needles collected at the edge of the Malvési uranium facility (SW France) exhibit enhanced activities of actinides and some decay products (uranium, americium, plutonium, (230)Th, (226)Ra) compared to a remote site. These enhanced activities resulted from the release of U via smokestacks and passive release from former artificial ponds located inside the nuclear site. Enhanced activities are also observed in selected produce (wheat, lettuce, fruits) sampled from the edge of the site. However, excess actinides measured in wheat grains in 2007 are inconsistent with the activities and the uranium ratio measured in the soils. This result suggests that the studied annual crops were contaminated mainly through the short-term release of airborne actinides, and that other transfer pathways, such as, uptake through the roots or adhesion of soil particles, were negligible.

Isotopic evidence of natural uranium and spent fuel uranium releases into the environment.

Uranium and plutonium isotopes were measured in soils, sediments and waters in an area subject to the past and present discharges from the uranium conversion plant of Malvési (France). The isotopes (236)U and (239)Pu are well known activation products of uranium and they prove to be powerful tracers of spent fuel releases in soils and sediments. On the other hand (234)U and (238)U activities measured in waters can be used to distinguish between releases and background uranium sources. Such findings contribute to improve the monitoring of the actinides releases by nuclear fuel facilities (mining sites, conversion, enrichment and fuel plants, reprocessing plants).

Mus spretus SEG/Pas mice resist virulent Yersinia pestis, under multigenic control.

Laboratory mice are well known to be highly susceptible to virulent strains of Yersinia pestis in experimental models of bubonic plague. We have found that Mus spretus-derived SEG/Pas (SEG) mice are exceptionally resistant to virulent CO92 and 6/69 wild type strains. Upon subcutaneous injection of 10(2) colony-forming units (CFU), 90% of females and 68% of males survived, compared with only an 8% survival rate for both male and female C57BL/6 mice. Furthermore, half of the SEG mice survived a challenge of up to 10(7) CFU. The time required for mortality was similar between B6 and SEG, suggesting that survival is dependent on early rather than late processes. The analysis of 322 backcross mice identified three significant quantitative trait loci (QTLs) on chromosomes 3, 4 and 6, with dominant SEG protective alleles. Each QTL increased the survival rate by approximately 20%. The three QTLs function additively, thereby accounting for 67% of the difference between the parental phenotypes. Mice heterozygous for the three QTLs were just as resistant as SEG mice to Y. pestis challenge. The SEG strain therefore offers an invaluable opportunity to unravel mechanisms and underlying genetic factors of resistance against Y. pestis infection.

Accuracy of continuous subcutaneous glucose monitoring with the GlucoDay in type 1 diabetic patients treated by subcutaneous insulin infusion during exercise of low versus high intensity.

AIM: The GlucoDay allows continuous glucose monitoring by subcutaneous microdialysis in sedentary conditions. To validate it when glycaemia may undergo rapid and dramatic changes, we investigated its accuracy during two exercise sessions with markedly different glucose disposal rates. METHODS: Nine male diabetic patients, aged 32-61, treated by insulin pumps, first underwent a standard maximal exercise-test designed for determining the maximal oxygen consumption and the first ventilatory threshold (Vt1). Then two 30 min steady-state workloads at 15% below and 15% above the Vt1 were performed in random order with the GlucoDay, and measurement of CHO oxidation rates was made by indirect calorimetry. RESULTS: CHO oxidation during exercise at +15% Vt1 was higher (+943.5 mg/min, ie +45.5%, P<0.01) than during exercise at -15% Vt1 No hypoglycaemia occurred. Due to breakages of 39% of subcutaneous probes, eleven steady-state sessions in 7 subjects allowed to compare 141 paired glucose (sensor vs. venous) determinations. The Clarke error grid situates 92.9% of glucose values within the A zone and 6.4% within the B zone, while only one pair of values (0.7%) falls in the D zone. Venous glucose tended to decrease more rapidly than sensor glucose during exercise. Bland-Altman plots evidence for a few cases of underestimation of venous glucose at high intensity. CONCLUSIONS: This study showed satisfactory accuracy of the GlucoDay during exercise. A slight lag time in sensor values likely explains a few discrepancies that do not appear as clinically meaningful. Reduction of probe fragility and confirmed sensor accuracy in hypoglycaemia would further support applicability of GlucoDay at exercise.

Glycerol and NEFA kinetics in long-term fasting king penguins: phase II versus phase III.

In spontaneously fasting birds such as penguins, below a body mass threshold corresponding to the phase II-phase III transition, a metabolic and hormonal shift occurs and feeding behaviour is stimulated ('refeeding signal'). The major aim of this study was to determine whether a decrease in non-esterified fatty acid (NEFA) release from adipose tissue could be a component of this signal. Lipolytic fluxes and primary triacylglycerol:fatty acid (TAG:FA) cycling were determined in vivo in breeding, fasting king penguins (Aptenodytes patagonicus) using continuous infusions of 2-[3H]glycerol and 1-[14C]palmitate under field conditions. In phase II (after approximately 8 days of fasting, large fat stores, body protein spared, N=8), the rate of appearance (R(a)) of glycerol and of NEFA were 5.7+/-0.8 and 10.5+/-0.4 micromol kg(-1) min(-1), respectively, and the percentage of primary TAG:FA cycling was 41+/-7%. In phase III (after approximately 25 days of fasting, fat stores reduced by fourfold, increased body protein catabolism, N=9), R(a) glycerol kg(-1) body mass remained unchanged, whereas R(a) glycerol kg(-1) fat mass and R(a) NEFA kg(-1) body mass were increased by 2.8-fold and 1.5-fold, respectively. Increased R(a) glycerol kg(-1) fat mass was possibly the result of a 3.5-fold increase in circulating glucagon, the increased R(a) NEFA kg(-1) body mass being attributable to decreased primary TAG:FA cycling. Thus, triggering of the refeeding signal that redirects the behavior of fasting, incubating penguins from incubation towards the search for food after entrance into phase III cannot be ascribed to a reduction in lipolytic fluxes and NEFA availability.

Delivery of multiple epitopes by recombinant detoxified adenylate cyclase of Bordetella pertussis induces protective antiviral immunity.

CyaA, the adenylate cyclase toxin from Bordetella pertussis, can deliver its N-terminal catalytic domain into the cytosol of a large number of eukaryotic cells and particularly into professional antigen-presenting cells. We have previously identified within the primary structure of CyaA several permissive sites at which insertion of peptides does not alter the ability of the toxin to enter cells. This property has been exploited to design recombinant CyaA toxoids capable of delivering major histocompatibility complex (MHC) class I-restricted CD8(+) T-cell epitopes into antigen-presenting cells and to induce specific CD8(+) cytotoxic T-lymphocyte (CTL) responses in vivo. Here we have explored the capacity of the CyaA vector carrying several different CD8(+) T-cell epitopes to prime multiple CTL responses. The model vaccine consisted of a polyepitope made of three CTL epitopes from lymphocytic choriomeningitis virus (LCMV), the V3 region of human immunodeficiency virus gp120, and chicken ovalbumin, inserted at three different sites of the catalytic domain of genetically detoxified CyaA. Each of these epitopes was processed on delivery by CyaA and presented in vitro to specific T-cell hybridomas. Immunization of mice by CyaA toxoids carrying the polyepitope lead to the induction of specific CTL responses for each of the three epitopes, as well as to protection against a lethal viral challenge. Moreover, mice primed against the vector by mock CyaA or a recombinant toxoid were still able to develop strong CTL responses after subsequent immunization with a recombinant CyaA carrying a foreign CD8(+) CTL epitope. These results highlight the potency of the adenylate cyclase vector for induction of protective CTL responses with multiple specificity and/or broad MHC restriction.

Refeeding signal in fasting-incubating king penguins: changes in behavior and egg temperature.

This study is directed toward understanding the process of feeding stimulation ("refeeding signal") that has been suggested to operate below a body mass threshold or critical metabolic status in spontaneously fasting birds. Behavior and egg temperature (T(egg)) were continuously monitored by video monitoring and biotelemetry, respectively, in fasting-incubating king penguins kept in a pen to prevent relief by the partner until spontaneous egg abandonment. Penned birds fasted 10 days more and lost 1.2 kg more than birds relieved normally by their partner, abandoning the egg about 1 wk after reaching a critical body mass. Definitive egg abandonment was preceded by transitory abandonments of progressively increasing duration during which time the birds went further and further away from their egg. There were marked interindividual differences but on average transitory abandonments began 36 +/- 5 h before the definitive abandonment and were paralleled by resumption of display songs signaling the readiness of the bird to depart for feeding. T(egg) was maintained at around 35.7 degrees C during normal incubation but significantly decreased the last 2 days before egg abandonment. These changes are interpreted as reflecting a stimulation to refeed at a threshold body mass corresponding to a critical fat store depletion. Thus the fasting-incubating king penguin appears to be an interesting animal model for understanding the long-term metabolic control of feeding behavior in relation to energy status.

Lipid composition of erythrocytes and thrombocytes of a subantarctic seabird, the king penguin.

Phospholipid (PL) compositions and fatty acid (FA) patterns of PL were determined in the erythrocytes and blood thrombocytes of a seabird, the king penguin, living in the subantarctic area and feeding on prey rich in n-3 polyunsaturated FA. Results were compared between birds in three different physiological states (breeding and molting adults, chicks) to those reported for other birds. In erythrocytes, the ratios of cholesterol to PL and of sphingomyelin to phosphatidylcholine (PC) were lower than in other birds. The PL distribution was similar to those previously reported in the hen and pigeon. In contrast to other birds, cardiolipin levels were unexpectedly high (4%). Very long chain n-3 FA were abundant (13-27%) in phosphatidylethanolamine (PE), phosphatidylserine and PC, probably in relation to the natural diet of these birds. Among n-3 FA, 22:6n-3 was the most abundant in all PL (2-20%), whereas the highest levels of arachidonic acid were observed in PE (14%). In thrombocytes, the PL distribution and FA composition of the main PL (PC, PE) differed from those of erythrocytes, and in particular, levels of n-3 FA (9-12%) were 1.5-2 times lower. The highest levels of arachidonic acid were found in phosphatidylinositol (24%). The lipid profile of penguin erythrocytes could contribute to the efficiency of blood circulation and oxygen delivery in microvascular beds, thus favoring diving capacity of these animals. Our observations do not support the hypothesis of a common origin of avian thrombocytes and erythrocytes.

Lipid composition of blood platelets and erythrocytes of southern elephant seal (Mirounga leonina) and antarctic fur seal (Arctocephalus gazella).

Erythrocyte and blood platelet phospholipid compositions were studied in three elephant seals and two fur seals, two species of marine mammals living in the Subantarctic region feeding on preys rich in (n-3) polyunsaturated fatty acids. Results were compared with those reported for related species and humans. In erythrocytes, the phospholipid (PL) and cholesterol (CHOL) contents were lower in pinnipeds than in humans. Phosphatidylcholine (PC) levels were higher in elephant seals than in fur seals, with a reverse trend for phosphatidylethanolamine (PE) and phosphatidylserine (PS). Both species had lower SM/PC ratios and PE plasmalogen concentrations than human. Erythrocytes were richer in (n-3) fatty acids (FA) in pinnipeds than in humans. In platelets, the PL content was lower and the CHOL content higher in elephant seals than in humans or in other phocid seal species studied to date. The SM/PC ratio was much higher than in other seal species or in man. In both species, the proportion of PE plasmalogens was higher in platelets than in erythrocytes. PL were more saturated in elephant seals than in fur seals. These results suggest that the erythrocytes and platelets of wild marine mammals may prove useful models to study the influence of dietary lipids on the structure and hemostatic function of these cells.

In vivo induction of CTL responses by recombinant adenylate cyclase of Bordetella pertussis carrying multiple copies of a viral CD8(+) T-cell epitope.

Bordetella pertussis adenylate cyclase toxin (ACT) is one of the few known protein toxins penetrating directly into the cytosol of target cells across their cytoplasmic membrane without the need for endocytosis. This capacity of ACT was recently exploited for in vivo delivery of single viral CD8(+) T-epitopes into MHC class I-presenting cells and induction of protective antiviral cytotoxic T-cell (CTL) responses. Here, we have explored the potential of the cell-invasive adenylate cyclase domain of the toxin to deliver larger antigens by evaluating the epitope-specific CTL responses induced by constructs bearing one to four copies of the CD8(+) T-epitope from the nucleoprotein of the lymphocytic choriomeningitis virus. The increase in the number of copies of the epitope was accompanied by a moderate decrease of the specific cell invasiveness of the ACT protein and did not lead to further enhancement of the level of induced epitope-specific CTL cells in mice, as compared to ACT with a single copy of the epitope. These results demonstrate the capacity of ACT to deliver larger heterologous antigens comprising several epitopes for antigenic presentation in vivo.

Therapy of murine tumors with recombinant Bordetella pertussis adenylate cyclase carrying a cytotoxic T cell epitope.

Bordetella pertussis secretes an invasive adenylate cyclase toxin, CyaA, that is able to deliver its N-terminal catalytic domain into the cytosol of eukaryotic target cells directly through the cytoplasmic membrane. We have shown previously that recombinant CyaA can be used to deliver viral CD8+ T cell epitopes to the MHC-class I presentation pathway to trigger specific CTL responses in vivo. In the present study, we show that mice immunized with a detoxified but still invasive CyaA carrying a CD8+ T cell epitope of OVA developed strong epitope-specific CTL responses, which kill tumor cells expressing this Ag. Treating mice with this recombinant molecule after the graft of melanoma cells expressing OVA induced a strong survival advantage compared with control animals. To our knowledge, this study represents the first demonstration that a nonreplicative and nontoxic vector carrying a single CTL epitope can stimulate efficient protective and therapeutic antitumor immunity.

Charge-dependent translocation of Bordetella pertussis adenylate cyclase toxin into eukaryotic cells: implication for the in vivo delivery of CD8(+) T cell epitopes into antigen-presenting cells.

Bordetella pertussis secretes a calmodulin-activated adenylate cyclase toxin, CyaA, that is able to deliver its N-terminal catalytic domain (400-aa residues) into the cytosol of eukaryotic target cells, directly through the cytoplasmic membrane. We have previously shown that CyaA can be used as a vehicle to deliver T cell epitopes, inserted within the catalytic domain of the toxin, into antigen-presenting cells and can trigger specific class I-restricted CD8(+) cytotoxic T cell responses in vivo. Here, we constructed a series of recombinant toxins harboring at the same insertion site various peptide sequences of 11-25 amino acids, corresponding to defined CD8(+) T cell epitopes and differing in the charge of the inserted sequence. We show that inserted peptide sequences containing net negative charges (-1 or -2) decreased or completely blocked (charge of -4) the internalization of the toxin into target cells in vitro and abolished the induction of cytotoxic T cell responses in vivo. The blocking of translocation due to the inserted acidic sequences can be relieved by appropriate mutations in the flanking region of CyaA that counterbalance the inserted charges. Our data indicate that (i) the electrostatic charge of the peptides inserted within the catalytic domain of CyaA is critical for its translocation into eukaryotic cells and (ii) the delivery of T cell epitopes into the cytosol of antigen-presenting cells by recombinant CyaA toxins is essential for the in vivo stimulation of specific cytotoxic T cells. These findings will help to engineer improved recombinant CyaA vectors able to stimulate more efficiently cellular immunity.

Determination of 226Ra in mineral drinking waters by alpha liquid scintillation with rejection of beta-gamma emitters.

The radiotoxicity of radium isotopes (especially the long-half-life 226Ra) requires their monitoring in drinking waters or nuclear wastes. We studied the applicability of the PERALS method of detection (photon electron rejecting alpha liquid scintillation) for radium measurement. This method combines alpha liquid scintillation with pulse shape analysis for beta rejection and specific chemical extractants included in the scintillating cocktail. Radium is separated by an extractive-scintillator cocktail called RADAEX containing 2-methyl-2-heptylnonanoic acid (HMHN) and dicyclohexano-21-crown-7 (Cy(2)21C7) as extractant molecules. The variation of the radium extraction has been studied relative to pH, salt concentrations, anion and cation effects, and the volume ratio between aqueous and organic phases. The main parameter affecting the radium extraction in mineral drinking water is its complexation by inorganic anions, especially sulfate. Due to the lack of thermodynamic data, some complexation constants had to be determined. For instance, the value reported in this paper for radium sulfate (log beta = 2.58 +/- 0.22) is in good agreement with that from the literature. The knowledge of complexation constants allows the determination of radium extraction recovery for any solution when the inorganic anion concentrations had been measured by capillary zone electrophoresis. The detection limit for this technique is found to be equal to 0.006 Bq.L-1 using only 6 mL of sample solution for analysis. Several French mineral waters have been studied and the results compared with determinations of uranium and thorium concentrations by ICPMS and time-resolved laser induced fluorescence (TRLIF).