Increasing the cAMP concentration during in vitro maturation of pig oocytes improves cumulus maturation and subsequent fertilization in vitro.

Porcine IVF faces various problems such as incomplete cytoplasmic maturation of the oocyte and polyspermy. Previous studies proved the importance of cAMP in regulating nuclear and cytoplasmic maturation of oocytes. This study investigated the effect of the cAMP-modulating agents 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cAMP sodium salt (dbcAMP) on several parameters during in vitro production of porcine embryos. First, we wanted to see if oocyte collection in IBMX could meiotically arrest oocytes and, as such, improve synchronization of nuclear and cytoplasmic maturation. To this end, cumulus-oocyte complexes (COCs) were collected from gilts in HEPES-buffered Tyrode balanced salt solution medium with 0.5-mM IBMX or without IBMX. At the end of oocyte collection, the effect of IBMX on chromatin configuration was evaluated. However, no differences could be observed in nuclear configuration between IBMX- and IBMX+ oocytes (P > 0.05). Second, we added dbcAMP during IVM to improve cytoplasmic maturation and evaluated cumulus expansion (lack of adhesion), a disintegrin and metalloproteinase with thrombospondin-like repeats (ADAMTS-1) levels in cumulus cells, fertilization, and blastocyst rates. Cumulus-oocyte complexes were matured in modified North Carolina State University medium 37 with or without 1-mM dbcAMP. Frozen-thawed, epididymal, boar spermatozoa were used for IVF. After IVF, presumed zygotes were cultured for 7 days in North Carolina State University medium 23. Penetration rate decreased in dbcAMP+ (57.3%) compared with dbcAMP- (67.8%), but the polyspermy rate also decreased (43.3% vs. 53.4%, respectively) leading to an increased normal fertilization rate (56.7% vs. 46.6%, respectively; P < 0.05). Only 7.2% of the COCs showed adhesion in dbcAMP+ which was lower than 15.7% in dbcAMP- (P < 0.05) probably because of an upregulation of the ADAMTS-1 protein by dbcAMP. When the adherent oocytes were removed during maturation, no difference could be detected between the blastocyst rate of dbcAMP- and dbcAMP+ (17.1% and 21.0% on Day 7, respectively; P > 0.05). In conclusion, the use of IBMX during collection did not cause a meiotic arrest. Using dbcAMP during IVM caused a greater normal fertilization rate, a lower rate of adherent COCs during IVM, higher levels of ADAMTS-1 in cumulus cells, and an equal blastocyst rate after screening out adherent COCs. These findings contribute to a better understanding of cAMP involvement in porcine oocyte maturation and provide a basis to develop an improved system with less polyspermy and higher blastocyst rates.

Current knowledge on the transport and fate of spermatozoa in the reproductive tract of the bitch.

Canine sperm transport, distribution, storage and detachment is a complex, dynamic and highly regulated process. Transport of sperm within the bitch's reproductive tract is rapid and is influenced by the method of semen deposition (natural mating or artificial insemination) and by the timing of breeding in relation to the day of ovulation. The fertile lifespan of spermatozoa in the reproductive tract of the bitch is considerably longer than in most other domestic species, and the main sperm reservoirs appear to be the uterine crypts and the distal part of the uterotubal junction, where spermatozoa attach by their heads to uterine epithelium. While several in vitro studies demonstrated prolonged motility and viability of canine spermatozoa after coincubation with uterine tube explants, spermatozoal storage has not been documented in the canine uterine tube isthmus or ampulla in vivo. Several factors, including exposure to progesterone, solubilized zona pellucida proteins and post-ovulation uterine tube fluid, appear to trigger membrane events resulting in capacitation-like changes with subsequent motility pattern changes (transitional and hyperactivated) that are associated with sperm detachment. After mating or insemination, a normal low-magnitude post-mating uterine inflammatory response occurs, evidenced by an influx of polymorphonuclear neutrophils (PMNs), increased uterine contractions and an increased uterine artery blood flow. Recently, it was also shown that normal dogs with cystic endometrial hyperplasia develop a more significant endometritis, show fewer mating-induced uterine contractions, a decreased ability of spermatozoa to bind to uterine explants in vitro and a slower uterine clearance after mating.

Peripartum changes in orexigenic and anorexigenic hormones in relation to back fat thickness and feeding strategy of sows.

Highly prolific sows often experience peripartum hypophagia, resulting in decreased production rate. Leptin, ghrelin, and resistin are known as feed intake-regulating hormones in many species, but it is yet unknown how feeding strategy and body condition will affect these hormones around parturition in sows. In the present study, a total of 63 sows, parity 2 to 7 were divided over 2 treatment groups which were fed either restricted (RESTRICT) or ad libitum (ADLIB) during the peripartum period (day 106 of gestation until day 7 of lactation). Within each treatment group, sows were assigned to 1 of 3 body condition groups based on back fat thickness at day 106 of gestation: <18 mm (LEAN), between 18 and 22 mm (MODERATE), and >22 mm (FAT). Postprandial blood samples were taken on days 107, 109, and 112 of gestation and on days 1, 3, and 5 of lactation. With RIA, leptin, ghrelin, and resistin of each sample were analyzed. For both leptin and resistin, the hormonal profile gradually increased throughout the peripartum period (P < 0.001), whereas ghrelin peaked on day 109 of gestation compared with day 107 of gestation and day 1 of lactation. Other time points were intermediate between those two (P < 0.001). The peripartum profile of leptin was significantly higher for FAT sows than for the 2 other condition groups. No effect of body condition on ghrelin and resistin concentrations was observed. None of the 3 measured hormones were affected by feeding strategy. In conclusion, during the peripartum period feed intake of sows did not affect leptin, ghrelin, or resistin profiles. Leptin was the only hormone investigated that reflected body condition. Although body condition and late gestation feed intake have been previously described as risk factors for peripartum hypophagia, they did not induce hypophagia in any of the sows or affect the profile of the observed feed intake-regulating hormones during the peripartum period.

MO-F-BRB-04: Fast Estimation of Secondary Particle Therapy Dose Using a Modified Track Repeating Method.

PURPOSE: Charged particle beams are of great interest, because they can achieve highly conformal radiation dose distributions. Despite this, some scattered radiation is inevitably present outside of the target volumes, and is of concern because of risks such as radiogenic cancer. Accurately calculating the secondary dose in regions far from the target volume is very difficult due to extremely low particle fluence and the effect of heterogeneities on particle ranges, making calculations possible only with CPU-week long Monte Carlo runs. By using a modified track repeating method, we demonstrate fast and accurate estimation of secondary dose appropriate for clinical use. METHODS: Primary and secondary particle track databases (including protons, electrons, photons, neutrons, and positrons) were generated with the Geant4 Monte Carlo toolkit. Several new strategies were developed or employed to improve the performance of non- primary particle propagation, including: (1) processing the databases such that only primary tracks producing deep penetrating photons or neutrons were kept and particles falling below transport thresholds were discarded, (2) a search algorithm that can locate a sub-track for a given energy in constant time, (3) multiplying photon and neutron tracks during propagation and scoring using particle 'splitting1. RESULTS: Performance and accuracy were benchmarked against full Monte Carlo calculations (Geant4 and FLUKA). Filtering out tracks that did not produce deep penetrating photons or neutrons did not affect the accuracy of the secondary dose calculation. Preliminary performance analysis indicated 60- 100X speed up over Fluka and 700-1000X speed up over Geant4 with well maintained accuracy. CONCLUSIONS: Estimation of secondary dose from particle therapy has so far been largely an academic exercise. This method for fast estimation of secondary dose brings patient / plan specific information within reach, allowing clinicians to make informed decisions on the potential long-term risks associated with specific dose delivery plans. Partial funding from NSF grant CBET-0853157.

Comparison of oral versus parenteral iron supplementation on the health and productivity of piglets.

The aim of this study was to compare oral iron supplementation in feed with a standard iron injection and assess the impact on the health and productivity of pigs. The reliability of a quick test (HemoCue) for the measurement of blood haemoglobin concentration was also investigated. Three pig herds, with a total of 88 sows and their piglets, were included in the study. Piglets in treatment group O were fed a special iron-rich feed on days 2 to 4, 5 to 7 and 8 to 12 of lactation using a specially designed feeding device. Piglets in group IM received 200 mg of an iron dextran complex intramuscularly at three days of age. Haemoglobin concentrations and performance of the piglets were compared between groups. The mean haemoglobin concentrations in pigs at weaning were 131.4 and 116.4 g/l for pigs in groups O and IM, respectively (P<0.01). Daily weight gain (253.9 v 248.8 g/day) and piglet mortality (11.4 v 12.2 per cent) were slightly better in group O than in group IM (P>0.05).

Dose-response effect of fish oil substitution in parturition feed on erythrocyte membrane characteristics and sow performance.

The present study aimed to investigate whether n-3 polyunsaturated fatty acids (PUFA) incorporate into erythrocyte membranes of peripartal sows in a dose-responsive manner and whether the altered fatty acid profile affects the cell membrane characteristics. At day 109 of gestation (day 0), 51 sows were divided into five treatment groups. Each group received a diet with a different ratio of fish oil to pork lard for nine consecutive days. Blood samples were taken at day 0 and 10 days later. The fatty acid profile of erythrocytes was determined, as well as the osmotic fragility and oxidative stability of erythrocytes. Thiobarbituric acid reactive substances (TBARS) and ferric reducing ability of plasma (FRAP) were determined in plasma samples. Finally, reproductive and performance parameters of both sows and piglets were recorded until weaning. Supplementation of fish oil during the peripartal period changed the fatty acid profile of erythrocyte membranes in a dose-responsive manner. Although the n-3 PUFA content of erythrocyte membranes increased with increasing amounts of fish oil in the diet, no significant effect on erythrocyte osmotic fragility could be recorded. In contrast, oxidative stability of erythrocytes decreased linearly with increasing amounts of fish oil in the diet. Similarly, both TBARS and FRAP linearly increased with increasing percentages of fish oil in the diet. Neither piglet nor sow performance was influenced by dietary treatments, except for a decrease of both piglet survival and weaning weight with increasing quantities of fish oil supplemented. It is concluded that changes in dietary lipid sources can affect the membrane's fatty acid profile within days, and mainly influences oxidative stability of the cells.

Validation of ATP luminometry for rapid and accurate titration of Mycoplasma hyopneumoniae in Friis medium and a comparison with the color changing units assay.

Limited reports are available on the growth response of Mycoplasma hyopneumoniae in Friis medium and the routinely used color changing units (CCU) assay has not yet been profoundly compared with other titration methods. Firstly, growth kinetics of 7 diverse M. hyopneumoniae isolates were followed by ATP luminometry in five Friis medium batches. Secondly, results of the CCU and ATP assays were compared hereby evaluating the methods. Growth curves of all isolates had log, stationary and senescence phases, and reached similar maximal titres when cultured in the same batch of Friis medium. Doubling times (Tds) of the isolates grown in slowly shaken cultures varied between 4.8 and 7.8 h. Maximal titres, Tds, growth phase in which the phenol red indicator turned from red to yellow due to acidification by mycoplasmal metabolism, and the length of the stationary phase varied depending on the Friis medium batch. The effect of static vs. shaking culture conditions on the Td depended on the isolate. ATP and CCU assays obtained similar growth curves, but when maximal levels were reached the CCU titre dropped earlier than the ATP titre. During log phase, CCU and ATP titres were strongly linearly linked. We developed a model enabling transformation of ATP into CCU titres or vice versa. The calculated amount of ATP per CCU (1.77 amol ATP/ml) indicated that the CCU assay likely underestimates the actual cell concentration. When titres were determined as means of 3 measurements, the ATP assay was 7 times more accurate and had 11-fold lower outliers than the CCU assay. Unlike the CCU assay, luminometry only requires one measurement to obtain sufficient accuracy. It was concluded that the ATP assay constitutes a valuable robust alternative for reproducible real-time titre assessment of freshly grown M. hyopneumoniae cultures. It is faster, more accurate and time, work and cost efficient compared to the CCU assay. The assay is preferred to better standardise and describe M. hyopneumoniae cultures used in various experiments.

Short-term exposure to hydrogen peroxide during oocyte maturation improves bovine embryo development.

Recent studies have shown that short-term exposure of oocytes to a stressor such as hydrostatic pressure or osmotic stress might induce stress tolerance in embryos. The aim of the present study was to investigate the consequences of short-term hydrogen peroxide (H(2)O(2)) exposure to bovine in vitro matured cumulus-oocyte complexes (COCs) on subsequent preimplantation embryo development and apoptosis. In the first experiment, mature COCs were incubated in H(2)O(2) at concentrations ranging between 0.01 and 100 micromol/l, and subsequently fertilized and cultured. Oocyte incubation with 50-100 micromol/l of H(2)O(2) resulted in a significantly higher blastocyst yield (47.3%) in comparison with control medium (31.8%), while apoptotic cell ratio was inversely related with H(2)O(2) concentration. In the second experiment, we showed that the stress tolerance after H(2)O(2) exposure was not mediated by increased glutathione content in treated oocytes nor by enhanced fertilization or penetration. Further research should concentrate on the potential role of players that have been associated with stress tolerance in somatic cell lines.

In vivo virulence of Mycoplasma hyopneumoniae isolates does not correlate with in vitro adhesion assessed by a microtitre plate adherence assay.

AIMS: Adherence of Mycoplasma hyopneumoniae to the ciliated epithelial cells of the porcine respiratory tract is considered an important first step in the pathogenesis of enzootic pneumonia. It was the aim of this study to verify the usefulness of in vitro adhesion as a virulence marker. METHODS AND RESULTS: Adherence capacity to immobilized cilia from porcine tracheal epithelial cells of three low, two moderately and two highly virulent M. hyopneumoniae field isolates was determined by a microtitre plate adherence assay. CONCLUSIONS: No significant differences between the isolates were demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that mechanisms other than adherence might be responsible for the observed differences in virulence of these field isolates or that the in vitro assay does not adequately reproduce in vivo adherence conditions.

Dorsoproximal proximal phalanx osteochondral fragmentation in 117 Warmblood horses.

The objective of the present study was to determine clinical and arthroscopic characteristics associated with dorsoproximal proximal phalanx (P1) fragments in Warmblood horses, as well as to examine their histopathological appearance. One hundred sixty-eight fragments were removed from 150 fetlocks of 117 Warmblood horses. Details of signalment and results of clinical examination were collected prior to surgery. After arthroscopic fragment removal and joint evaluation for synovial and/or cartilage abnormalities, the fragments were measured and evaluated histopathologically. The vast majority of the fragments (95.2%) were found medially, without predilection for front or hind limbs. In 10% of the joints, more than one fragment was present. The mean size of the fragments was 6.8 +/- 2.6 mm. Only eight horses presented fetlock-related lameness. Horses of seven years of age and older (OR = 13.32; p = 0.033) and the presence of more than one fragment (OR = 11.12; p = 0.016) were significantly associated with lameness. Arthroscopic evaluation revealed one or more abnormalities in 50.7% of the joints. On histopathology, osteochondral fragments presented as a bony center covered with smooth hyaline cartilage on one side and some fibrous tissue on the other side. No clear histopathological signs were indicating precisely their origin. In Warmblood horses with dorsoproximal P1 fragments, the age (seven years and older) and the presence of more than one fragment in a fetlock significantly increased the risk of lameness. The osteochondral dorsoproximal P1 fragments could be defined as a developmental orthopaedic disease.

Influence of postpartum cloprostenol treatment in sows on subsequent reproductive performance under field conditions.

During the previous decade several studies focused on postpartum treatment with prostaglandin for improvement of reproductive performance in sows. The aim of the study was to investigate the effect of administration of a prostaglandin F(2 alpha) (PGF(2 alpha)) analogue in sows within 24-48 h after farrowing on sow and litter performance. In five commercial farms, the sows were randomly assigned to either treatment A (2 ml cloprostenol, Planate) or treatment B (2 ml physiological saline solution, i.m.). Fifteen per cent of all sows were at random selected for progesterone analysis. Litter performance was assessed by measuring pre-weaning mortality and average daily weight gain (ADG). Sow performance was assessed by measuring weaning-to-oestrus interval (WOI), the percentage of sows returning to oestrus and litter size during subsequent farrowing. Administration of a PGF(2 alpha) analogue within 24-48 h postpartum had no effect on the rate of progesterone decline measured over 24 h compared with that of the controls. Litter performance and WOI were not affected by treatment. The subsequent litter size in sows of parity seven and more showed a significant difference of 1.98 piglets (p < 0.01) between both groups, to the benefit of the cloprostenol group. In conclusion, administration of a synthetic PGF(2 alpha) analogue, cloprostenol, within 24-48 h after farrowing improved litter size at next farrowing in older (>or=7 parity) sows.

Prevention of oxidative damage that contributes to the loss of bioenergetic capacity in ageing skin.

Skin ageing is a complex biological process related to a decline in physiological and biochemical performance. A decline in the mitochondrial energy production is a feature of ageing at the cellular level. This is partially attributed to excessive production of reactive oxygen species such as superoxide and hydrogen peroxide in aged individuals. We have investigated the effect of (glyc)oxidative stress on two biochemical targets relevant for the energy metabolism of the skin. First, we showed an age dependent decline in the activity of the hydrogen peroxide detoxifying antioxidant catalase in stratum corneum on a chronically sun-exposed site. Furthermore catalase was sensitive to peroxynitrite-induced in vitro inactivation. Catalase mimetics as well as peroxynitrite scavengers are proposed to maintain hydrogen peroxide detoxification pathways. The second target was creatine kinase, an enzyme that controls the creatine-creatine phosphate shuttle. Creatine kinase lost activity after in vitro glycation by methylglyoxal. This activity loss could be prevented by antiglycation actives. These data suggest that biomolecules involved in energy homeostasis become damaged by different sources of stress. Actives specifically selected for optimal protection against these stress situations will decrease skin vulnerability and prevent the premature loss of skin function.

Effect of non-esterified fatty acids on bovine theca cell steroidogenesis and proliferation in vitro.

Elevated serum non-esterified fatty acid (NEFA) levels associated with a negative energy balance (NEB) may affect ovarian function and hence reproductive performance in high-yielding dairy cows. We have investigated the individual and combined effects of the three major NEFAs on bovine theca cell proliferation and steroidogenesis in vitro. Theca cells from healthy large follicles (>8 mm) obtained from slaughterhouse ovaries were cultured in serum free medium in the presence of 0, 50, 150 and 200 microM of palmitic acid (PA; C16:0); 0, 50, 150 and 250 microM of stearic acid (SA; C18:0); and/or 0, 50, 150 and 250 microM of oleic acid (OA; C18:1). Progesterone and androstenedione concentrations were measured in spent medium after 48 h of culture and cell numbers were determined spectrophotometrically per culture well. Cell viability was assessed by annexin-V FITC/propidium iodide staining. Only the treatment with 200 microM of PA inhibited cell proliferation (P<0.001) when tested individually, both of the mixtures tested (M1=100 microM of PA, 130 microM of SA and 140 microM of OA; M2=200 microM PA, 260 microM of SA and 280 microM of OA) reduced cell numbers (P<0.001). Progesterone and androstenedione production, both per well and per 10(4) cells, were not affected by any of the treatments, with the exception of M2. This mixture reduced progesterone production per well and per 10(4) cells (P<0.05). The effects observed were most likely caused by the cytotoxic action of the NEFAs, as demonstrated by the increased percentage of early apoptotic (M1) and late apoptotic/necrotic cells (M1 and M2) in the combination treatments (P<0.05). When combined, elevated physiological concentrations of PA, SA and OA can modulate theca cell proliferation and steroidogenesis in vitro by reducing theca cell viability. These NEFAs may be one of the mediators through which NEB compromises ovarian functioning and thus fertility in high-yielding dairy cows.

Effect of non-esterified fatty acids on bovine granulosa cell steroidogenesis and proliferation in vitro.

In high-yielding dairy cows, the negative energy balance (NEB) during the first weeks post partum may influence dominant follicle growth and steroidogenesis. Since non-esterified fatty acid (NEFA) concentrations are elevated during NEB and are shown to be toxic for several cell types, we investigated the individual and combined effects of the three main NEFA's on granulosa cell proliferation and steroidogenesis in vitro. Granulosa cells from large follicles were cultured for two days in serum free medium in the presence of palmitic (C16:0) (PA), stearic (C18:0) (SA) and/or oleic acid (C18:1) (OA). Addition of 150, 300 or 500 microM of PA and SA inhibited cell proliferation (P<0.05) while OA only elicited such an effect at 500 microM (P<0.01). In the combination treatment (150 microM of each fatty acid), cell numbers were also reduced (P<0.01). These inhibitory effects on cell number are partly due to the induction of apoptosis by these NEFA's, as was demonstrated by annexin V-FITC/propidium iodide staining of the granulosa cells. Oestradiol-17beta production was stimulated by all doses of PA, by 300 and 500 microM of SA and by 500 microM of OA (P<0.05). Combined treatment with 150 microM of each fatty acid also stimulated oestradiol-17beta production per 10(4) cells (P<0.05). We can conclude that PA, SA and to a lesser degree OA modulate granulosa cell proliferation and steroidogenesis in vitro. These effects may be involved in the occurrence of ovarian dysfunction during the postpartum period in high-yielding dairy cows.

MYRRHA, a Pb-Bi experimental ADS: specific approach to radiation protection aspects.

Since 1998, SCK*CEN, in partnership with IBA s.a. and many European research laboratories, is designing a multipurpose accelerator driven system (ADS) for Research and Development (R&D) applications-MYRRHA-and is conducting an associated R&D support programme. MYRRHA is an ADS under development at Mol in Belgium and is aiming to serve as a basis for the European experimental ADS to provide protons and neutrons for various R&D applications. It consists of a proton accelerator delivering a 350 MeV x 5 mA proton beam to a liquid Pb-Bi spallation target that in turn couples to a Pb-Bi cooled, subcritical fast core. In the first stage, the project focuses mainly on demonstration of the ADS concept, safety research on sub-critical systems and nuclear waste transmutation studies. In a later stage, the device will also be dedicated to research on structural materials, nuclear fuel, liquid metal technology and associated aspects, and on sub-critical reactor physics. Subsequently, it will be used for research on applications such as radioisotope production. A first preliminary conceptual design file of MYRRHA was completed by the end of 2001 and has been reviewed by an International Technical Guidance Committee, which concluded that there are no show stoppers in the project and even though some topics such as the safety studies and the fuel qualification need to be addressed more deeply before concluding it. In this paper, we are reporting on the state-of-the art of the MYRRHA project at the beginning of 2004 and in particular on the radiation shielding assessment and the radiation protection particular aspects through a remote handling operation approach in order to minimise the personnel exposure to radiation.

Cigarette smoke-induced lipid peroxidation in human skin and its inhibition by topically applied antioxidants.

Cigarette smoke, whether indirect or direct stream, is an environmental pollutant which presents an increasing health problem. In order to determine damage to human skin at the biochemical level, volar forearms were exposed to cigarette smoke for fifteen minutes and then assayed for the presence of stratum corneum lipid peroxides. A time-dependent increase was observed over a 24-hour post-exposure period. At 24 h, the average baseline level of lipid peroxides was 14.9 nmol/unit area of skin as compared to 32.0 nmol/unit area of skin for the smoke-exposed arms. In addition, when topical antioxidants were pre-applied to the skin and then exposed to cigarette smoke, an average decrease of 40.9% in lipid peroxide values was observed. These data demonstrate that peroxidation was induced in human skin by cigarette smoke and subsequently inhibited by the presence of antioxidants.

Human keratinocytes respond to osmotic stress by p38 map kinase regulated induction of HSP70 and HSP27.

Human skin is exposed to an environment that varies in humidity from 100 to 0%, leading to seasonal variations in the condition of the skin. Exposure to a low humidity environment creates an osmotic gradient across the stratum corneum, which is known to modulate cutaneous barrier function. Heat shock proteins protect against stress-induced destabilization of proteins. We investigated whether osmotic shock (sorbitol) induced a heat shock protein response in normal human keratinocytes, and used heat shock as a positive control. Both heat shock and osmotic stress (200 and 300 mM sorbitol) clearly induced heat shock proteins 70 and 27 mRNA levels. The induction of heat shock protein 70 mRNA levels by osmotic stress peaked at 16 h and persisted until 24 h, whereas upregulation of heat shock protein 70 mRNA levels by heat peaked at 2 h and returned to baseline levels by 6 h. Sorbitol also increased heat shock protein 70 levels in a concentration-dependent manner. The kinetics of heat shock protein 27 mRNA induction by osmotic stress and heat were similar with peak induction at 6 h. The mitogen activated protein kinase family of proteins plays an important part in the coordination of gene responses to various stress conditions. We have demonstrated that the p38 mitogen activated protein kinase was strongly activated by 200 mM and 300 mM sorbitol. The specific p38 mitogen activated protein kinase inhibitor PD169316 almost completely blocked heat shock protein 70 mRNA induction by 200 mM and 300 mM sorbitol and completely suppressed heat shock protein 27 mRNA induction with 200 mM sorbitol. PD169316 also counteracted upregulation of heat shock protein 70 levels by sorbitol. These data indicate that keratinocytes respond to osmotic stress by p38 mitogen activated protein kinase regulated induction of heat shock proteins. This molecular pathway may be relevant for the mechanisms regulating the response of human skin to variations in environmental humidity.

Correlation between in vitro cyclic adenosine monophosphate phosphodiesterase inhibition and in vivo anti-inflammatory effect.

We tested the hypothesis that topical adenosine monophosphate phosphodiesterase (cAMP PDE) inhibitors are anti-inflammatory. These can be shown by a correlation between PDE inhibitory and anti-inflammatory function of a series of known PDE inhibitors. The effect of various cAMP PDE inhibitors on PDEs isolated from HaCaT cells was first investigated. These compounds were then tested as anti-irritants against topical 8% Balsam of Peru. A direct correlation was observed between the in vitro EC(50) values for PDE inhibition and the in vivo anti-inflammatory potential with a correlation coefficient of r = 0.79. These results stress the value of PDE inhibitors as anti-inflammatory agents in topical use, and also demonstrate that the in vitro PDE assay can be used to predict in vivo anti-inflammatory potential.

Successful separation of apoptosis and necrosis pathways in HaCaT keratinocyte cells induced by UVB irradiation.

UVB irradiation can induce apoptotic, necrotic, and differentiation pathways in normal human keratinocytes. The present study was undertaken to determine at what dose of UVB each of these pathways is induced and whether these pathways are distinct or overlapping. We have observed that UVB induces fragmentation of DNA in human HaCaT keratinocytes, in a bimodal manner. Low doses of UVB, 5-20 mJ/cm2, increase the levels of apoptosis as shown by increased levels of fragmented DNA, Fas, PARP, and FasL protein, and the number of apoptotic cells as assessed by FACS analysis. At higher doses of UVB (20 and 30 mJ/cm2) the number of apoptotic cells becomes reduced, as does the amount of Fas, PARP, and FasL protein. At these higher doses, cell viability is decreased as measured by DNA synthesis (BrdU labeling) neutral red uptake, which represents an increasing necrotic phenotype. Expression of markers of keratinocyte differentiation, involucrin, keratin K1, and keratin K10, are also observed to decrease with increasing UVB dose. These changes are accompanied by a further increase in DNA fragmentation. We conclude that low doses of UVB (5-20 mJ/cm2) induced an apoptotic pathway, whereas increasing doses (greater than 20 mJ/cm2) of UVB produce a direct necrotic effect and inhibit terminal differentiation.

[Extensive mycotic coronary aneurysm detected by echocardiography. Apropos of a case]. / Volumineux anévrisme mycotique coronaire de découverte échographique. A propos d'un cas.

The authors report the case of a large mycotic right coronary aneurysm detected at echocardiography in a 45 year old patient with AIDS. Although emergency surgery was planned, the patient died of rupture of the aneurysm with cardiogenic shock and sudden pericardial tamponade. This case underlines the diagnostic value of echocardiography, by the transthoracic approach for para-cardiac masses and with the transoesophageal probe for accurate localisation and demonstration of the coronary origin. In this case, the CT scan was less useful than transthoracic echocardiography. Coronary angiography confirmed the strongly suggestive echocardiographic diagnosis and helped decide management strategy. Atheromatous coronary aneurysms may be treated by stenting but mycotic aneurysms require surgical management.