Use of a real-time microbial air sampler for operational cleanroom monitoring.

A sampler that detects and counts viable particles in the air of cleanrooms in real-time was studied. It was found that when the sampler was used to monitor airborne particles dispersed from a number of materials used in cleanrooms, including garments, gloves, and skin, the number of viable particles dispersed from these materials was greater than anticipated. It was concluded that a substantial proportion of these viables were of a non-microbiological origin. When the sampler was used to monitor a non-unidirectional airflow cleanroom occupied by personnel wearing cleanroom garments, it was found that the airborne viable concentrations were unrealistically high and variable in comparison to microbe-carrying particles simultaneously measured with efficient microbial air samplers. These results confirmed previously reported ones obtained from a different real-time sampler. When the real-time sampler was used in a workstation within the same cleanroom, the recorded viables gave results that suggest that the sampler may provide an effective airborne monitoring method, but more investigations are required. LAY ABSTRACT: The airborne concentrations measured by a real-time microbial air sampler within an operational, non-unidirectional airflow cleanroom were found to be unrealistically high due to a substantial numbers of particles of non-microbiological origin. These particles, which resulted in false-positive microbial counts, were found to be associated with a number of materials used in cleanrooms. When the sampler was used within a cleanroom workstation, the counts appeared to be more realistic and suggest that this type of real-time airborne microbial counter may provide a useful monitoring method in such workstations, but further investigations are required.

Comparative genomic hybridization and pathological findings in atypical teratoid/rhabdoid tumour of the central nervous system.

The atypical teratoid/rhabdoid tumour (AT/RT) is an uncommon tumour of the central nervous system in children, characterized by the presence of a rhabdoid cell component associated with variable combinations of primitive neuroectodermal tumour, mesenchymal and epithelial differentiation. Immunohistochemistry reveals a complex pattern of antigen expression and cytogenetic studies have demonstrated losses from chromosome 22. We have performed comparative genomic hybridization (CGH) on paraffin-embedded material from three cases of AT/RT. Two cases showed losses from chromosome 22 associated with other chromosome imbalances including losses from 1p in both cases. The third case demonstrated a loss from 8p as the sole abnormality. While monosomy or deletion from chromosome 22 is a useful diagnostic marker for AT/RT, it is not present in all cases. The variation in cytogenetic patterns reported for this tumour type raises the possibility that different genetic pathways may underlie this tumour phenotype and warrants the further definition of the cytogenetic spectrum for this rare tumour.

CD80-mediated induction of immunostimulation in two ocular melanoma cell lines is augmented by interferon-gamma.

Although the transfection of the T-cell costimulatory molecule CD80 cDNA into human tumours can augment their immunogenicity in vitro, its expression alone is ineffective in many tumour systems. We evaluated the influence of CD80 expression on the immunostimulatory activity of ocular melanoma cell lines and determined whether IFN-gamma could enhance the effect. Two ocular melanoma cell lines were transfected with CD80 cDNA. The immunostimulatory capacity of the CD80+ transfectants was determined by their ability to stimulate the proliferation of allogeneic peripheral blood mononuclear cells (PBMC). The influence of additional accessory molecules on PBMC proliferation was assessed by pre-treating the CD80 transfectants with IFN-gamma. The CD80+ transfectants induced proliferation of allogeneic PBMC. IFN-gamma treatment of the tumour cells induced upregulated expression of MHC class I, de novo expression of MHC class II and CD54, and enhanced the ability of the CD80+ transfectants to stimulate PBMC proliferation. CD4+ T cells were not required for the proliferative response against untreated CD80+ tumour cells but were necessary for the augmentation of proliferation observed following IFN-gamma treatment. CD80+ ocular melanoma cells possess immunostimulatory potential which is augmented by IFN-gamma induced upregulation of cell surface molecules. Further studies on the role of costimulatory molecules in inducing anti-tumour immunity in ocular melanoma may help to define new strategies for application of immunotherapeutic approaches to treat this aggressive disease.

Pitfalls in the use of thyrotropin concentration as a first-line thyroid-function test.

Measurement of thyrotropin concentration alone as a first-line thyroid-function test fails to indicate hypopituitarism in a number of patients. Using a combination of thyrotropin and thyroxine assays, we analysed 56,000 tests for a population of 471,000 over 12 months. 15 patients with clinically unsuspected hypopituitarism were detected, indicating that the occurrence of hypopituitarism might be underestimated.

Influence of selenium supplements on the post-traumatic alterations of the thyroid axis: a placebo-controlled trial.

OBJECTIVE: To investigate whether early selenium (Se) supplementation can modify the post-traumatic alterations of thyroid hormone metabolism, since the first week after trauma is characterised by low plasma Se and negative Se balances. DESIGN: Prospective, placebo-controlled randomised supplementation trial. SETTING: Surgical ICU in a tertiary university hospital. PATIENTS: Thirty-one critically ill trauma patients aged 42 +/- 16 years (mean +/- SD), with severe multiple injury (Injury Severity Score 30 +/- 7). INTERVENTION: Supplementation during the first 5 days after injury with either Se or placebo. The selenium group was further randomised to receive daily 500 microg Se, with or without 150 mg alpha-tocopherol (AT) and 13 mg zinc supplements. The placebo group received the vehicle. Circulating Se, AT, zinc, and thyroid hormones were determined on D0 (= day 0, admission), D1, D2, D5, D10, and D20. RESULTS: Plasma Se, low on D0, normalised from D1 in the selenium group; total T4 and T3 increased more and faster after D2 (P = 0.04 and 0.08), reverse T3 rising less between D0 and D2 (P = 0.05). CONCLUSIONS: Selenium supplements increased the circulating Se levels. Supplementation was associated with modest changes in thyroid hormones, with an earlier normalisation of T4 and reverse T3 plasma levels. The addition of AT and zinc did not produce any additional change.

Hypermanganesemia in long-term intravenous nutrition and chronic liver disease.

BACKGROUND: Hypermanganesemia and cholestatic liver disease are both recognized complications of long-term IV nutrition. Manganese is primarily excreted in bile, and recent studies have indicated that manganese toxicity may play a role in the pathogenesis of IV nutrition-associated cholestasis. METHODS: Whole blood and plasma manganese concentrations were measured in patients receiving long-term home IV nutrition (HIN, n = 30). Whole blood manganese concentrations also were measured in patients with chronic liver disease (CLD, n = 10) and control subjects (n = 10). RESULTS: Whole blood manganese concentrations of all CLD patients were within the reference interval (73 to 210 nmol/L) and were not different from those of the control group (151 +/- 44 nmol/L, CLD vs 155 +/- 35 nmol/L, control; not significant), despite the presence of cholestasis. In contrast, whole blood manganese concentration was increased (>210 nmol/L) in 26 patients, and plasma manganese concentration increased (>23 nmol/L) in 23 of the patients receiving HIN. None of the patients exhibited neurologic signs of manganese toxicity. There was no correlation between whole blood manganese concentrations and markers of cholestasis, IV manganese intake, or duration of HIN. However, plasma manganese concentration correlated both with average weekly IV manganese intake (r = .44, p = .02) and with gamma-glutamyl transferase (r = .43, p = .02) and alkaline phosphatase activities (r = .55, p = .003). CONCLUSIONS: Cholestatic liver disease does not appear to contribute to increased whole blood manganese concentrations in patients not receiving HIN. Plasma manganese concentrations in patients receiving HIN reflect recent manganese exposure and impaired excretion where cholestasis is present. The lack of relationship between plasma and whole blood manganese concentrations suggests that factors other than manganese intake and excretion affect intracellular concentrations.

Trace element supplementation modulates pulmonary infection rates after major burns: a double-blind, placebo-controlled trial.

Infections remain the leading cause of death after major burns. Trace elements are involved in immunity and burn patients suffer acute trace element depletion after injury. In a previous nonrandomized study, trace element supplementation was associated with increased leukocyte counts and shortened hospital stays. This randomized, placebo-controlled trial studied clinical and immune effects of trace element supplements. Twenty patients, aged 40 +/- 16 y (mean +/- SD), burned on 48 +/- 17% of their body surfaces, were studied for 30 d after injury. They consumed either standard trace element intakes plus supplements (40.4 micromol Cu, 2.9 micromol Se, and 406 micromol Zn; group TE) or standard trace element intakes plus placebo (20 micromol Cu, 0.4 micromol Se, and 100 micromol Zn; group C) for 8 d. Demographic data were similar for both groups. Mean plasma copper and zinc concentrations were below normal until days 20 and 15, respectively (NS). Plasma selenium remained normal for group TE but decreased for group C (P < 0.05 on days 1 and 5). Total leukocyte counts tended to be higher in group TE because of higher neutrophil counts. Proliferation to mitogens was depressed compared with healthy control subjects (NS). The number of infections per patient was significantly (P < 0.05) lower in group TE (1.9 +/- 0.9) than in group C (3.1 +/- 1.1) because of fewer pulmonary infections. Early trace element supplementation appears beneficial after major burns; it was associated with a significant decrease in the number of bronchopneumonia infections and with a shorter hospital stay when data were normalized for burn size.

Myotomal slow muscle function of rainbow trout Oncorhynchus mykiss during steady swimming.

Strain and activity patterns were determined during slow steady swimming (tailbeat frequency 1.5-2.5 Hz) at three locations on the body in the slow myotomal muscle of rainbow trout Oncorhynchus mykiss using sonomicrometry and electromyography. Strain was independent of tailbeat frequency over the range studied and increased significantly from +/-3.3 % l0 at 0.35BL to +/-6 % at 0.65BL, where l0 is muscle resting length and BL is total body length. Muscle activation occurred significantly later in the strain cycle at 0.35BL (phase shift 59 degrees) than at 0.65BL (30 degrees), and the duration of activity was significantly longer (211 degrees at 0.35BL and 181 degrees at 0.65BL). These results differ from those of previous studies. The results have been used to simulate in vivo activity in isolated muscle preparations using the work loop technique. Preparations from all three locations generated net positive power under in vivo conditions, but the negative power component increased from head to tail. Both kinematically, and in the way its muscle functions to generate hydrodynamic thrust, the rainbow trout appears to be intermediate between anguilliform swimmers such as the eel, which generate thrust along their entire body length, and carangiform fish (e.g. saithe Pollachius virens), which generate thrust primarily at the tail blade.

A search for immunoglobulins inhibiting gonadal cell steroidogenesis in premature ovarian failure.

Premature ovarian failure (POF) may be caused by the action of circulating gonadotrophin receptor-blocking antibodies. Luteinizing hormone (LH)-stimulated testosterone production from mouse Leydig cells and follicle stimulating hormone (FSH)-stimulated oestradiol production from immature rat Sertoli cells were therefore studied in the presence of protein-G purified immunoglobulin G (IgG) samples from control subjects (n = 9), infertile women with elevated early follicular phase FSH levels but otherwise normal menstrual cycles (n = 10), and patients with POF (n = 10) or Graves' disease (n = 10). A saturating and subsaturating (78% for LH; 60% for FSH) dose of each hormone was chosen for study. A commercial preparation of human IgG (Sigma IgG, 0.75 mg/ml) employed as negative control had no effect on basal or gonadotrophin-stimulated steroidogenesis. In its presence, saturating doses of LH (2 IU/l) and FSH (20 IU/l) gave rise to 11.2 +/- 0.8 (n = 7) and 25.1 +/- 5.8 (n = 8) fold increases in steroid secretion. IgG (0.75 mg/ml) had no effect in the four groups on LH-stimulated testosterone outputs using a saturating (2 IU/l) or subsaturating (1 IU/l) dose of hormone. For example, LH (2 IU/l)-stimulated testosterone production was 94% (83-96 median; interquartile range) and 88% (81-99) of the Sigma IgG control for control and POF groups respectively. However, four out of nine IgG samples from the normal subjects (mean +/- SEM = 86 +/- 6%), two out of 10 of the high FSH group (77 +/- 4%), five out of 10 with Graves' disease (86 +/- 3%) and six out of 10 with POF (76 +/- 6%) gave rise to LH (2 IU/l)-stimulated testosterone outputs which were lower (P < 0.05) than that of Sigma IgG control. Using the identical set of patients and an IgG concentration of 0.25 mg/ml, the FSH-stimulated oestradiol outputs of the four groups were similar when using either the saturating (20 IU/l) or subsaturating (5 IU/l) dose of the hormone. Thus, the percentage of FSH (20 IU/l)-stimulated oestradiol production of the Sigma IgG control was 81 (66-89 median, interquartile range) and 50 (38-84) for control and POF groups respectively. However, once again individual patients had inhibitory IgGs such that four out of nine (controls), three out of 10 (high FSH group), four out of 10 (Graves' disease) and six out of 10 (POF patients) inhibited (P < 0.05) FSH (20 IU/l)-stimulated oestradiol secretion by 52 +/- 9 (mean +/- SEM), 44 +/- 7, 52 +/- 6 and 41 +/- 6% respectively. Of the patients with inhibitory IgGs the extent of inhibition of gonadotrophin-stimulated steroid secretion was similar between the groups. In conclusion, there is little evidence to suggest that immunoglobulins blocking gonadotrophin receptors are a mechanism for POF in a large proportion of women suffering from this condition.

Volume-sensitive chloride currents in primary cultures of human fetal vas deferens epithelial cells.

Using the patch-clamp technique, we have identified a large, outwardly rectifying, Cl--selective whole-cell current in primary cultures of human vas deferens epithelial cells. Whole-cell currents were time- and voltage-dependent and displayed inactivation following depolarising pulses >/= 60 mV. Currents were equally permeable to bromide (PBr/PCl = 1.05 +/- 0.04), iodide (PI/PCl = 1. 06 +/- 0.07) and Cl-, but significantly less permeable to gluconate (PGluc /PCl = 0.23 +/- 0.03). Currents spontaneously increased with time after establishing a whole-cell recording, but could be inhibited by exposure to a hypertonic bath solution which reduced inward currents by 68 +/- 4%. Subsequent exposure of the cells to a hypotonic bath solution led to a 418 +/- 110% increase in inward current, indicating that these currents are regulated by osmolarity. 4,4'-Diisothiocyanatostilbene-2,2'-disulphonic acid (100 microM) produced a rapid and reversible voltage-dependent block (60 +/- 5% and 10 +/- 7% inhibition of current, measured at +/- 60 mV, respectively). Dideoxyforskolin (50 microM) also reduced the volume-sensitive Cl- current, but with a much slower time course, by 41 +/- 13% and 32 +/- 16% (measured at +/- 60 mV, respectively). Tamoxifen (10 microM) had no effect on the whole-cell Cl- current. These results suggest that vas deferens epithelial cells possess a volume-sensitive Cl- conductance which has biophysical and pharmacological properties broadly similar to volume-sensitive Cl- currents previously described in a variety of cell types.

Two barium binding sites on a maxi K+ channel from human vas deferens epithelial cells.

Using the patch clamp technique, we have investigated the blockade of maxi-K+ channels present on vas deferens epithelial cells by extracellular Ba2+. With symmetrical 140 mM K+ solutions, Ba2+ produced discrete blocking events consisting of both long closings of seconds duration (slow block) and fast closings of milliseconds duration (flickering block). Kinetic analysis showed that flickering block occurred according to an "open channel blocking" scheme and was eliminated by reducing external K+ to 4.5 mM. Slow block showed a complex voltage-dependence. At potentials between -20 mV and 20 mV, blockade was voltage-dependent; at potentials greater than 20 mV, blockade was voltage-independent, but markedly sensitive to the extracellular K+ concentration. These data reveal that the vas deferens maxi-K+ channel has two Ba2+ binding sites accessible from the extracellular side. Site one is located at the cytoplasmic side of the gating region and binding to this site causes flickering block. Site two is located close to the extracellular mouth of the channel and binding to this site causes slow block.

In-vitro biopotency and glycoform distribution of recombinant human follicle stimulating hormone (Org 32489), Metrodin and Metrodin-HP.

In this study the in-vitro biopotency and glycoform distribution of human recombinant follicle stimulating hormone (FSH, Org 32489) has been assessed. The biopotency of recombinant FSH was studied using animal (rat Sertoli) and human (granulosa-lutein) cell models. Recombinant FSH, as measured in the rat Sertoli cell assay, was more potent than the urinary preparations Metrodin, Metrodin-HP and IS 70/45 with half maximal stimulation (ED50; mean +/- SEM, n > 3) occurring at 2.2 +/- 0.5 IU/I (recombinant FSH), 4.7 +/- 1.1 IU/I (Metrodin), 13.2 +/- 0.7 IU/I (Metrodin-HP) and 6.4 +/- 0.3 IU/I (IS 70/45); the pituitary preparation IRP 83/575 had an ED50 of 10.4 +/- 0.1 IU/I. Using human granulosa-lutein cells, cultured for up to 4 days in the absence of exogenous steroid precursors, recombinant FSH was either without effect (three out of five patients) or inhibited both oestradiol and progesterone secretion. FSH (83/575) was without effect on oestradiol with preparations from any of the patients but slightly stimulated (134 +/- 8%; mean +/- SEM, P < 0.05) progesterone production at the highest dose (80 IU/I). The distribution of FSH isoforms, assessed by polyclonal radioimmunoassay, following chromatofocusing over the ranges pH < 3.5 and pH 3.5-7.0 respectively was recombinant FSH, 12.4 and 87.6%; Metrodin, 19.8 and 80.2%; Metrodin-HP, 50.2 and 49.8%; IS 70/45, 15.0 and 85.0%; IS 83/575, 70.9 and 29.1%. All glycoforms were pI < 7.0 for the five preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

cAMP-activated chloride channels in a CFTR-transfected pancreatic adenocarcinoma-derived cell line, pANS6.

Pancreatic adenocarcinoma cell lines rarely express the CFTR gene, despite the high levels of CFTR protein that are present in primary pancreatic duct cells. We have attempted to generate a non-CF pancreatic adenocarcinoma cell line that stably produces high levels of CFTR mRNA and protein by transfecting a vector containing the CFTR cDNA, driven by a strong mammalian promoter, into the poorly differentiated pancreatic adenocarcinoma cell line, Panc-1. The pANS6 pancreatic duct cell line expresses substantial levels of CFTR mRNA, but little CFTR protein. Despite this we were able to detect low conductance chloride channels in 40% of patches, stimulated with cAMP, that have similar biophysical properties to CFTR.

Characterization of DNASE I hypersensitive sites in the 120kb 5' to the CFTR gene.

The chromatin structure of 120kb of genomic DNA 5' to the CFTR gene has been analysed in a number of CFTR expressing and non-expressing cell types, including primary genital duct epithelial cells. Novel DNAse I hypersensitive sites have been observed at -79.5kb and -20.5kb 5' to the ATG translation start codon of the CFTR coding sequence. Neither of these sites appears to show strong correlation with CFTR expression in the cell types investigated, hence they are unlikely to reflect the sites of binding of the major CFTR tissue specific regulator(s). However, they may still play an important part in the complex series of events involved in the regulation of CFTR transcription.

Down-regulation of CFTR expression in an HT29-derived cell line by stable antisense RNA production.

In order to examine the interactions of the cAMP activated CFTR chloride ion channel with other chloride ion channels present in epithelial cells we have generated a model based on antisense mRNA down regulation of CFTR expression. The HT29-derived epithelial cell line CJC4-1 has integrated a plasmid that is constitutively expressing CFTR exons 1-6 in the antisense orientation, from the MoMULV LTR. This cell line shows a reduction in cAMP-activated chloride efflux, as measured by iodide efflux assay, in comparison to the parental HT29 cell line and a sense transfected control.

Molecular analysis of the ovine cystic fibrosis transmembrane conductance regulator gene.

There is a need for a large-animal model to investigate the etiology and biology of cystic fibrosis (CF) lung disease and to study potential therapies. The development and electrophysiology of the sheep airway have been shown to exhibit close functional parallels with the human airway, particularly with respect to the respiratory epithelium. We have cloned and sequenced the ovine cystic fibrosis transmembrane conductance regulator (CFTR) cDNA. It shows a high degree of conservation at the DNA coding and predicted polypeptide levels with human CFTR: at the nucleic acid level there is a 90% conservation (compared with 80% between human and mouse CFTR cDNA); at the polypeptide level, the degree of similarity is 95% (compared with 88% between human and mouse). Northern blot analysis and reverse transcription-PCR have shown that the patterns of expression of the ovine CFTR gene are very similar to those seen in humans. Further, the developmental expression of CFTR in the sheep is equivalent to that observed in humans. Thus, overall a CF sheep should show lung pathology similar to that of humans with CF.

Tuning in to fish swimming waves: body form, swimming mode and muscle function

Most fish species swim with lateral body undulations running from head to tail. These waves run more slowly than the waves of muscle activation causing them, reflecting the effect of the interaction between the fish's body and the reactive forces from the water. The coupling between both waves depends on the lateral body shape and on the mechanical properties of the tail. During steady swimming, the length of each myotomal muscle fibre varies cyclically. The phase relationship between the strain (muscle length change) cycle and the active period (when force is generated) determines the work output of the muscle. The muscle power is converted to thrust either directly by the bending body or almost exclusively by the tail, depending upon the body shape of the species and the swimming kinematics. We have compared the kinematics and muscle activity patterns from seven species of fish with different body forms and swimming modes and propose a model which yields a consistent pattern, with at least three extremes. Subtle tuning of the phase relationship between muscle strain and activation cycles can lead to major changes in the way muscles function in different swimming modes.