A mouse organotypic tissue culture model for autosomal recessive congenital ichthyosis.

BACKGROUND: Autosomal recessive congenital ichthyoses (ARCIs) are keratinization disorders caused by impaired skin barrier function. Mutations in the genes encoding the lipoxygenases 12R-LOX and eLOX-3 are the second most common cause of ARCIs. In recent years, human skin equivalents recapitulating the ARCI phenotype have been established. OBJECTIVES: To develop a murine organotypic tissue culture model for ARCI. METHODS: Epidermal keratinocytes were isolated from newborn 12R-LOX-deficient mice and cocultivated with mouse dermal fibroblasts embedded in a scaffold of native collagen type I. RESULTS: With this experimental set-up the keratinocytes formed a well-organized multilayered stratified epithelium resembling skin architecture in vivo. All epidermal layers were present and the keratinocytes within showed the characteristic morphological features. Markers for differentiation and maturation indicated regular epidermal morphogenesis. The major components of epidermal structures were expressed, and were obviously processed and assembled properly. In contrast to their wild-type counterparts, 12R-LOX-deficient skin equivalents showed abnormal vesicular structures in the upper epidermal layers correlating with altered lipid composition and increased transepidermal water loss, comparable with 12R-LOX-deficient mice. CONCLUSIONS: The mouse skin equivalents faithfully recapitulate the 12R-LOX-deficient phenotype observed in vivo, classifying them as appropriate in vitro models to study molecular mechanisms involved in the development of ARCI and to evaluate novel therapeutic agents. In contrast to existing human three-dimensional skin models, the generation of these murine models is not constrained by a limited supply of material and does not depend on in vitro expansion and/or genetic manipulations that could result in inadvertent genotypic and phenotypic alterations.

Activated sludge rheology: a critical review on data collection and modelling.

Rheological behaviour is an important fluid property that severely impacts its flow behaviour and many aspects related to this. In the case of activated sludge, the apparent viscosity has an influence on e.g. pumping, hydrodynamics, mass transfer rates, sludge-water separation (settling and filtration). It therefore is an important property related to process performance, including process economics. To account for this, rheological behaviour is being included in process design, necessitating its measurement. However, measurements and corresponding protocols in literature are quite diverse, leading to varying results and conclusions. In this paper, a vast amount of papers are critically reviewed with respect to this and important flaws are highlighted with respect to rheometer choice, rheometer settings and measurement protocol. The obtained rheograms from experimental efforts have frequently been used to build viscosity models. However, this is not that straightforward and a lot of errors can be detected with respect to good modelling practice, including fair model selection criteria, qualitative parameter estimations and proper model validation. These important steps are however recurrently violated, severely affecting the model reliability and predictive power. This is illustrated with several examples. In conclusion, dedicated research is required to improve the rheological measurements and the models derived from them. At this moment, there is no guidance with respect to proper rheological measurements. Moreover, the rheological models are not very trustworthy and remain very "black box". More insight in the physical background needs to be gained. A model-based approach with dedicated experimental data collection is the key to address this.

Genetic variations in fetal and maternal tumor necrosis factor-α and interleukin 10: is there an association with preterm birth or periventricular leucomalacia?

OBJECTIVE: The aim of the study was to identify whether tumor necrosis factor-α (TNF-α) (-308) and interleukin (IL)-10 (-1082; -819) genotypes were associated with preterm delivery and cystic periventricular leucomalacia (PVL). STUDY DESIGN: Venous blood, buccal swabs or cord blood were collected from mother/child pairs with infants born at term (200) or preterm (106) in the presence and absence of neonatal PVL and of premature infants with PVL (7). Extracted genomic DNA served as template for determination of IL-10 (-1082), IL-10 (-819) and TNF-α (-308) genotypes by allele-specific PCR. RESULT: No significant difference was observed in the frequencies of IL-10 (-1082), IL-10 (-819) and TNF-α (-308) genotypes in mothers or in children of term versus preterm deliveries with or without PVL. CONCLUSION: Maternal and infant IL-10 (-1082, -819) and TNF-α (-308) genotypes are not indicative for an increased risk of preterm birth or the development of PVL in premature newborns.

Principles of an enhanced MBR-process with mechanical cleaning.

Up to date, different physical and chemical cleaning protocols are necessary to limit membrane fouling in membrane bioreactors. This paper deals with a mechanical cleaning process, which aims at the avoidance of hypochlorite and other critical chemicals in MBR with submerged flat sheet modules. The process basically consists of the addition of plastic particles into the loop circulation within submerged membrane modules. Investigations of two pilot plants are presented: Pilot plant 1 is equipped with a 10 m(2) membrane module and operated with a translucent model suspension; pilot plant 2 is equipped with four 50 m(2) membrane modules and operated with pretreated sewage. Results of pilot plant 1 show that the establishment of a fluidised bed with regular particle distribution is possible for a variety of particles. Particles with maximum densities of 1.05 g/cm(3) and between 3 and 5 mm diameter form a stable fluidised bed almost regardless of activated sludge concentration, viscosity and reactor geometry. Particles with densities between 1.05 g/cm(3) and 1.2 g/cm(3) form a stable fluidised bed, if the velocity at the reactor bottom is sufficiently high. Activities within pilot plant 2 focused on plant optimisation and the development of an adequate particle retention system.

CXCR3+CD4+ T cells are enriched in inflamed kidneys and urine and provide a new biomarker for acute nephritis flares in systemic lupus erythematosus patients.

OBJECTIVE: The high frequency of CD4+ T cells in interstitial infiltrates of patients with lupus nephritis suggests a contribution of these cells to local pathogenesis. The aim of this study was to examine the role of CXCR3 and the chemokine CXCL10 in recruiting these cells into the kidney and to determine whether the infiltrating T cells could be monitored in the urine to provide a reliable biomarker for acute lupus nephritis. METHODS: The frequencies of CD3+ T cells, CXCR3+ cells, and CXCL10+ cells were determined by immunohistochemical and immunofluorescence analyses of kidney sections from 18 patients with lupus nephritis. The frequency of CXCR3+CD4+ T cells was determined by flow cytometry of peripheral blood and urine from 38 patients with systemic lupus erythematosus (SLE), and the values were compared with disease activity as determined by the Systemic Lupus Erythematosus Disease Activity Index. RESULTS: In renal biopsy tissues from patients with lupus nephritis, a mean of 63% of the infiltrating cells expressed CXCR3, approximately 60% of them were T cells, and the CXCR3+ cells colocalized with CXCL10-producing cells. In biopsy tissues from SLE patients with acute nephritis, approximately 50% of the urinary CD4+ T cells were CXCR3+, as compared with 22% in the peripheral blood, and the frequency of urinary CXCR3+CD4+ T cells correlated with disease activity. Moreover, the number of urinary CD4+ T cells reflected nephritis activity, and elevation above 800 CD4+ T cells per 100 ml of urine sharply delineated active from inactive nephritis. CONCLUSION: CXCR3+ T cells are recruited into the inflamed kidneys, are enriched in the urine, and are a valuable marker of nephritis activity in SLE. They also present a potential target for future therapies.

Virulence of low pathogenicity H7N2 avian influenza viruses from the Delmarva peninsula for broiler and leghorn chickens and turkeys.

The virulence of low pathogenicity (LP) type A H7N2 avian influenza virus (AIV) isolates recovered from chickens in Delaware and the eastern shore of Maryland in 2004 was evaluated. Three-week-old leghorn- and broiler-type chickens and turkeys were inoculated via the conjunctival sac with 10(3.5)-10(4.0) 50% embryo infections dose (EID50) of virus per bird with A/ chicken/Delaware/Viva/04, A/chicken/Delaware/Hobo/04, and A/chicken/Maryland/Minh Ma/04. In broilers, the viruses produced respiratory signs, airsacculitis, and microscopic lesions in the trachea and lung. In contrast, signs and lesions were less severe in turkeys, and they were rarely observed in specific-pathogen-free (SPF) leghorns. In broilers and SPF leghorns, AIV peaked on day 3 postinoculation (PI), based on virus isolation and real-time reverse transcription-polymerase chain reaction, and antigen capture testing. Infection in turkeys peaked on day 7 PI. Serum antibodies generally were detected earlier in broilers (day 7 PI) than in turkeys or SPF leghorns (day 14 PI) using agar gel immunodiffusion, hemagglutination-inhibition, and the enzyme-linked immunosorbent assay. A second trial was performed to further examine the disease susceptibility of the leghorn chicken given the comparatively mild responses noted in the first trial. A 10-fold higher dose of 10(4.5)-10(5.0)EID50 per chick given via the conjunctival sac was used. In addition, commercial-type leghorns were tested as were chicks from the SPF leghorn source. The higher AIV dose resulted in more rapid and consistent rates of infection and higher serum antibody responses in both types of leghorn chickens. However, as observed in the first trial, clinical signs and microscopic lesions in both types of leghorns were infrequent and very mild. These findings indicate leghorn-type chickens, which are commonly used for pathogenicity assessments because of their availability, may not be the most suitable host for evaluating the virulence potential of LP AIV.

Impact of colloidal and soluble organic material on membrane performance in membrane bioreactors for municipal wastewater treatment.

Two parallel membrane bioreactors (2 m3 each) were operated over a period of 2 years. Both pilots were optimised for nitrification, denitrification, and enhanced biological phosphorous elimination, treating identical municipal wastewater under comparable operating conditions. The only constructional difference between the pilots was the position of the denitrification zone (pre-denitrification in pilot 1 and post-denitrification in pilot 2). Despite identical modules and conditions, the two MBRs showed different permeabilities and fouling rates. The differences were not related to the denitrification scheme. In order to find an explanation for the different membrane performances, a one-year investigation was initiated and the membrane performance as well as the operating regime and characteristics of the activated sludge were closely studied. MLSS concentrations, solid retention time, loading rates, and filtration flux were found not to be responsible for the different performance of the submerged modules. These parameters were kept identical in the two pilot plants. Instead, the non-settable fraction of the sludges (soluble and colloidal material, i.e. polysaccharides, proteins and organic colloids) was found to impact fouling and to cause the difference in membrane performance between the two MBR. This fraction was analysed by spectrophotometric and size exclusion chromatography (SEC) methods. In a second step, the origin of these substances was investigated. The results point to microbiologically produced substances such as extracellular polymeric substances (EPS) or soluble microbial products (SMP).

Correlation between membrane fouling and soluble/colloidal organic substances in membrane bioreactors for municipal wastewater treatment.

Two similar membrane bioreactors of 2 m3 each were operated in parallel over two years under the same operational conditions, fed with the same municipal wastewater. The only process and operational difference between both pilot plants was the position of the denitrification zone (pre-denitrification in pilot 1 and post-denitrification in pilot 2). Despite parallel operation, the two MBRs exhibited different fouling rates and decreases in permeability. These differences could not be accounted for by MLSS concentrations, loading rates, or filtration flux. In a one-year investigation, soluble and colloidal organic material in the activated sludge of both MBR was regularly analysed by spectrophotometric and Size Exclusion Chromatography (SEC) methods. The larger organic molecules present in the sludge water phase (i.e. polysaccharides, proteins and organic colloids) originating from microbial activity (extracellular polymeric substances) were found to impact on the fouling and to explain the difference in membrane performance between the two MBR units. In both pilot plants, a linear relationship could be clearly demonstrated between the fouling rate of the membrane and the concentration of polysaccharides in the sludge water phase during a 5 month operational period at an SRT of 8 days.

Performance of a bioreactor with submerged membranes for aerobic treatment of municipal waste water.

Aerobic treatment of municipal waste water in a membrane bioreactor was studied for 535 d. Apart from sampling, sludge was retained completely by a submerged hollow fibre membrane with a pore-size of 0.2 microm. The pilot plant comprised an anoxic zone to enable denitrification. The maximum liquid hold-up of the plant was 3.9 m3. In this study the reactor performance and the stability of the process and the membrane capacity were investigated. A stable flux of 181 m(-2)h(-1) could be realised with a mean transmembrane pressure difference of 0.3bar with air-bubbling and backflushing the membrane and cleaning it in place every two months for one or two hours. For about 140d, a flux of 271 m(-2)h(-1) was achieved, but cleaning became necessary more often. The hydraulic retention time (HRT) varied between 10.4 and 15.6h. Accordingly the volumetric loading rate was between 1.1 and 1.7kg CODm(-3)d(-1). No inoculum was used. The mixed liquor suspended solids (MLSS) concentration gradually increased to 18-20g MLSSl(-1). The feed to microorganism (F/M) ratio varied according to the operation conditions but decreased against a value of 0.07 kg COD kg(-1) MLSSd(-1). Treatment performance was very stable and on a high level. The COD was reduced by 95%. Nitrification was complete and up to 82% of the total nitrogen could be denitrified.

Okadaic acid induces transcription of junB through a CCAAT box and NF-Y.

The shellfish toxin, okadaic acid (OA), is a potent tumor promoter that induces expression of the proto-oncogene junB in mouse keratinocyte 308 cells. Here we show, through deletion analysis of the junB promoter, that sequences near the TATA box conferred transcriptional induction by OA. Transient transfections of luciferase constructs bearing the junB promoter with single mutations in various cis elements demonstrated that a promoter containing a mutated CCAAT box could not be induced by OA. When this CCAAT box was inserted into a heterologous promoter construct, OA induction was dependent on an intact CCAAT box. Flanking cis elements located near the CCAAT box, although not required for OA inducibility, did play a role in the basal level of transcription. NF-Y was shown by EMSA to bind to the CCAAT box. OA induction from the junB CCAAT box was blocked by dominant negative NF-YA as well as the CCAAT box-dependent anticancer drug, ET-473. Expression of a lexA/NF-YA chimeric protein demonstrated that OA induction was dependent on the binding of NF-Y family members. These studies demonstrate that OA can mediate transcriptional activation of junB through the classical CCAAT box and that transcription factor NF-Y plays a functional role in the induction.

New genotypes in Fy(a-b-) individuals: nonsense mutations (Trp to stop) in the coding sequence of either FY A or FY B.

Duffy blood group antigens are carried on a glycoprotein that is predicted to pass through the erythrocyte membrane seven times and is a promiscuous chemokine receptor. The Fy(a- b-) phenotype is present in two-thirds of African-American Blacks but is rare in Caucasians. In Blacks, the phenotype is due to a non-functional GATA-1 motif in the FY B, which silences the gene in erythrocytes but not in other tissues, and these patients do not generally make anti-Fyb or anti-Fy3. We describe here the molecular analysis of FY in three unrelated Caucasians who were studied because they had strong anti-Fy3 in their serum. Each was found to have a point mutation that was predicted to change a tryptophan to a premature stop codon in the coding sequence. In one patient (patient 1), the nonsense mutation was at nucleotide 287 of the major transcript in FY A; in another (patient 2), it was at nucleotide 407 in the major transcript of FY B; and in a third (patient 3), it was at nucleotide 408 of the major transcript of FY A.

Increased ROS levels contribute to elevated transcription factor and MAP kinase activities in malignantly progressed mouse keratinocyte cell lines.

There is evidence that reactive oxygen species (ROS) are important mediators of tumor promotion and progression. The molecular mechanisms involved in ROS-mediated signaling, however, are unclear at present. Using ionizing radiation and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as model physical and chemical carcinogens, we have malignantly progressed 308 cells, a papilloma-producing mouse keratinocyte cell line, and investigated the molecular alterations in the progressed phenotypes. In this study, we demonstrate that both MNNG and radiation-progressed malignant variants showed elevated ROS levels that contributed to their proliferative capacity in vitro as well as in vivo. We found increased Erk-1/2 and p38 MAP kinase activities to be important components of ROS-mediated signaling. The pro-oxidant state also contributed to constitutive elevation of AP-1, NFkappaB and cAMP response element transactivation in the malignant phenotype. Our data provide evidence for a functional role of elevated ROS levels in tumor progression and implicate Erk-1/2 and p38 MAP kinase activation in the malignant progression of mouse keratinocytes.

Inhibition of p38 MAP kinase increases okadaic acid mediated AP-1 expression and DNA binding but has no effect on TRE dependent transcription.

By performing in vitro kinase assays we found in papilloma producing 308 mouse keratinocytes that okadaic acid elevated activities of extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinases (JNKs) and p38 mitogen-activated protein kinases (MAPKs). This okadaic acid mediated activation of MAP kinases correlated with increased AP-1 binding to a consensus TPA responsive element (TRE) and elevated TRE dependent transcription. To determine the role of p38 MAP kinases in these processes we employed the specific p38 MAP kinase inhibitor SB 203580. Using orthophosphate labeling we showed a decrease in phosphorylation of MAPK activated protein kinase-2 (MAPKAP-K2) indicating reduced activity of p38 MAPKs utilizing this kinase as substrate. In contrast, we found that SB 203580 raised activities of ERK-1/2 and JNKs. Electrophoretic mobility shift assays revealed an increase in TRE binding activity in response to SB 203580 most likely resulting from increased expression of the major TRE binding components JunD and FosB as indicated by Western blot analyses. Increased TRE DNA binding failed to lead to increased transactivation correlating with the inability of SB 203580 to increase phosphorylation of these AP-1 proteins. These data indicate that SB 203580 sensitive p38 MAP kinases are not involved in okadaic acid mediated increases in TRE DNA binding and transactivation.

Extracellular signal-regulated kinase 1/2-mediated phosphorylation of JunD and FosB is required for okadaic acid-induced activator protein 1 activation.

Previously, we reported that in papilloma-producing 308 mouse keratinocytes, the tumor promoter okadaic acid, a serine-threonine phosphatase inhibitor, increased binding of activator protein 1 (AP-1) to a consensus 12-O-tetradecanoylphorbol-13-acetate-responsive element (Rosenberger, S. F., and Bowden, G. T. (1996) Oncogene 12, 2301-2308). In this study, we investigated the correlation between AP-1 DNA binding and transactivation and examined molecular mechanisms involved in this process. Using a luciferase reporter driven by region -74 to +63 of the human collagenase gene, we demonstrated induction of AP-1-mediated transcription following okadaic acid treatment. By performing in vitro kinase assays, we found elevated activities of extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase. The ERK-1/2-specific inhibitor PD 98059 completely abrogated okadaic acid-induced AP-1 transactivation without altering AP-1 expression, DNA binding, or complex composition. Phosphorylation analyses indicated that inhibition of ERK-1/2 decreased okadaic acid-elevated phosphorylation of JunD and FosB. To further examine the role of JunD and FosB in okadaic acid-induced AP-1 transactivation, we generated fusion proteins of the DNA-binding domain of the yeast transcription factor Gal4 and the transactivation domain of either JunD or FosB. Cotransfection experiments of these constructs with a Gal4-luciferase reporter demonstrated that both JunD and FosB are required for okadaic acid-induced transcription. Treatment with PD 98059 reduced JunD/FosB-dependent transactivation, suggesting that ERK-1/2-mediated phosphorylation is a critical component in this process.

Okadaic acid stimulated TRE binding activity in a papilloma producing mouse keratinocyte cell line involves increased AP-1 expression.

The effects of the non-phorbol ester type tumor promoter okadaic acid, a serine-threonine phosphatase inhibitor, on activator protein 1 (AP-1) DNA binding activity were studied in papilloma producing 308 mouse keratinocytes. Okadaic acid increased AP-1 binding to a consensus TPA responsive element (TRE) within 2 h; maximum stimulation was observed at 6 h followed by a gradual decrease to basal levels within 24 h. Jun B, Jun D and Fos B proteins were identified as the major components of the AP-1 complex binding to the TRE element at 6 h. Inhibition of transcription with actinomycin D and inhibition of protein synthesis with cycloheximide abrogated the okadaic acid effect on AP-1 DNA binding, indicating that transcription and translation are required for okadaic acid increased TRE binding activity. Northern and Western blot analyses revealed a correlation between increased AP-1 binding activity and accumulation of jun B, jun D and fos B mRNAs and proteins. These data suggest increased AP-1 expression as principal mechanism of okadaic acid stimulated AP-1 activation in the mouse keratinocytes studied.

Prospective comparison of traditional and pharmacokinetic aminoglycoside dosing methods.

Aminoglycoside (gentamicin, tobramycin) dosage regimens and subsequent serum concentrations were compared in 30 patients treated initially using traditional physician-determined methods and then switched to a pharmacokinetic-based treatment program. Patients received more drug during the kinetic phase (median 5 mg/kg) than during the traditional phase (median 3.6 mg/kg) and achieved greater peak serum concentration (5.9 vs. 4.4 micrograms/ml). Seventy-three percent of kinetic peak values but only 27% of traditional peak values exceeded 5.0 micrograms/ml. Trough concentrations were comparable in both phases of study and no nephrotoxicity was observed. This pharmacokinetic-based management program achieved more consistently greater therapeutic peak concentrations and provided more individualized therapy than did physicians. The use of pharmacokinetic consultants may be of benefit in administering safely optimal aminoglycoside therapy.

Autoregulatory patterns in the arteriolar network of cat mesentery.

The purpose of this study was to examine the distribution and extent of autoregulatory behavior among arterioles in the arcade network of the cat mesentery. We found that 71% of 169 arterioles studied dilated and 63% showed blood flow autoregulation with arterial pressure reduction to 55-60 mm Hg. There appeared to be a direct relation between the degree of dilation and the degree of autoregulation when individual vessels were compared (r = 0.71). The dilation of individual arterioles was also correlated with the degree of autoregulation of other vessels in the same field (r = 0.51). To a lesser extent, the degree of dilation of individual arterioles was correlated with the degree of dilation of other vessels in the same field (r = 0.30). In comparing vessels which dilated, the largest average response was found in preparations where all vessels dilated. On a percentage basis the small arterioles located distally in the network dilated more than the large arterioles located proximally. In preparations having both reactive and nonreactive arterioles to pressure reduction, the nonreactive vessels were usually located geographically in proximity to each other. The flow distribution in the arteriolar network remained relatively stable on pressure reduction; the total tissue area perfused by a first-order arteriole shifting by 3% on the average.