Process development for a recombinant Chinese Hamster Ovary (CHO) cell line utilizing a metal induced and amplified metallothionein expression system

The suspension Chinese Hamster Ovary cell line, 13-10-302, utilizing the metallothionein (MT) expression system producing recombinant human growth hormone (hGH) was studied in a serum-free and cadmium-free medium at different fermentation scales and modes of operation. Initial experiments were carried out to optimize the concentration of metal addition to induce the MT promoter. Subsequently, the cultivation of the 13-10-302 cell line was scaled up from spinner flasks into bioreactors, and the cultivation duration was extended with fed-batch and perfusion strategies utilizing 180 muM zinc to induce the promoter controlling expression of recombinant hGH. It was shown that a fed-batch process could increase the maximum cell numbers twofold, from 3.3 to 6.3 x 10(6) cell/mL, over those obtained in normal batch fermentations, and this coupled with extended fermentation times resulted in a fourfold increase in final hGH titer, from 135 +/- 15 to 670 +/- 70 mg/L at a specific productivity q(hGH) value of 12 pg cell(-1)d(-1). The addition of sodium butyrate increased the specific productivity of hGH in cells to a value of approximately 48 pg cell(-1)d(-1), resulting in a final hGH titer of over a gram per liter during fed-batch runs. A BioSep acoustic cell recycler was used to retain the cells in the bioreactor during perfusion operation. It was necessary to maintain the specific feeding rates (SFR) above a value of 0.2 vvd/(10(6) cell/mL) to maintain the viability and productivity of the 13-10-302 cells; under these conditions the viable cell number increased to over 107 cell/mL and resulted in a volumetric productivity of over 120 mg(hGH) L(-1)d(-1). Process development described in this work demonstrates cultivation at various scales and sustained high levels of productivity under cadmium free condition in a CHO cell line utilizing an inducible metallothionein expression system. (C) 2004 Wiley Periodicals, Inc.

Mitochondria, endoplasmic reticulum and actin filament behavior after PDT with chloroaluminum phthalocyanine liposomal in HeLa cells

Photodynamic therapy (PDT) for cancer is a therapeutic modality in the treatment of tumors in which visible light is used to activate a photosensitizer. Cell membranes have been identified as an important intracellular target for singlet oxygen produced during the photochemical pathway. This study analyzed the cytotoxicity in specific cellular targets of a photosensitizer used in PDT in vitro. The photosensitizing effects of chloroaluminum phthalocyanine liposomal were studied on the mitochondria, cytoskeleton and endoplasmic reticulum of HeLa cells. Cells were irradiated with a diode laser working at 670 nm, energy density of 4.5 J/cm(2) and power density of 45 mW/cm(2). Fluorescence microscopic analysis of the mitochondria showed changes in membrane potential. After PDT treatment, the cytoskeleton and endoplasmic reticulum presented basic alterations in distribution. The combined effect of AlPHCl liposomal and red light in the HeLa cell line induced photodamage to the mitochondria, endoplasmic reticulum and actin filaments in the cytoskeleton. (c) 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Microextraction in packed sorbent for analysis of antidepressants in human plasma by liquid chromatography and spectrophotometric detection

A sensitive and reproducible method by microextraction packed sorbent and liquid chromatography with UV detection (MEPS/LC-UV) is described for the determination of new generation antidepressants (sertraline, mirtazapine, fluoxetine, citalopram and paroxetine) in human plasma samples. The MEPS variables, such as sample volume, pH, number of extraction cycles (draw-eject), and desorption conditions (solvent and solvent volume of elution) influenced the MEPS/LC efficiency significantly. Important factors in the optimization of MEPS efficiency, as well as washing steps and carryover effect are discussed. The analyses were carried out using small sample volumes (400 mu L.), and in a short time period (3 min for the entire sample preparation step). The MEPS/LC-UV method was shown to be linear at concentrations ranging from the limit of quantification (LOQ) to 1000 ng mL(-1). The LOQ values ranged from 10 to 25 ng mL(-1). The inter-day precision of the method presented coefficient of the variation ranging from 1.3% to 8.7%. On the basis of analytical validation, it is shown that the MEPS/LC-UV methodology is adequate for antidepressant analysis, from therapeutic to toxic levels. In order to evaluate the proposed method for clinical use, the MEPS/LC-UV method was applied to analysis of plasma samples from elderly depressed patients. (C) 2010 Elsevier B.V. All rights reserved.

Royal Australian and New Zealand College of Psychiatrists clinical practice guidelines for the treatment of schizophrenia and related disorders

Background: The Royal Australian and New Zealand College of Psychiatrists is co-ordinating the development of clinical practice guidelines (CPGs) in psychiatry, funded under the National Mental Health Strategy (Australia) and the New Zealand Health Funding Authority. This paper presents CPGs for schizophrenia and related disorders. Over the past decade schizophrenia has become more treatable than ever before. A new generation of drug therapies, a renaissance of psychological and psychosocial interventions and a first generation of reform within the specialist mental health system have combined to create an evidence-based climate of realistic optimism. Progressive neuroscientific advances hold out the strong possibility of more definitive biological treatments in the near future. However, this improved potential for better outcomes and quality of life for people with schizophrenia has not been translated into reality in Australia. The efficacy-effectiveness gap is wider for schizophrenia than any other serious medical disorder. Therapeutic nihilism, under-resourcing of services and a stalling of the service reform process, poor morale within specialist mental health services, a lack of broad-based recovery and life support programs, and a climate of tenacious stigma and consequent lack of concern for people with schizophrenia are the contributory causes for this failure to effectively treat. These guidelines therefore tackle only one element in the endeavour to reduce the impact of schizophrenia. They distil the current evidence-base and make recommendations based on the best available knowledge. Method: A comprehensive literature review (1990-2003) was conducted, including all Cochrane schizophrenia reviews and all relevant meta-analyses, and a number of recent international clinical practice guidelines were consulted. A series of drafts were refined by the expert committee and enhanced through a bi-national consultation process. Treatment recommendations: This guideline provides evidence-based recommendations for the management of schizophrenia by treatment type and by phase of illness. The essential features of the guidelines are: (i) Early detection and comprehensive treatment of first episode cases is a priority since the psychosocial and possibly the biological impact of illness can be minimized and outcome improved. An optimistic attitude on the part of health professionals is an essential ingredient from the outset and across all phases of illness. (ii) Comprehensive and sustained intervention should be assured during the initial 3-5 years following diagnosis since course of illness is strongly influenced by what occurs in this 'critical period'. Patients should not have to 'prove chronicity' before they gain consistent access and tenure to specialist mental health services. (iii) Antipsychotic medication is the cornerstone of treatment. These medicines have improved in quality and tolerability, yet should be used cautiously and in a more targeted manner than in the past. The treatment of choice for most patients is now the novel antipsychotic medications because of their superior tolerability and, in particular, the reduced risk of tardive dyskinesia. This is particularly so for the first episode patient where, due to superior tolerability, novel agents are the first, second and third line choice. These novel agents are nevertheless associated with potentially serious medium to long-term side-effects of their own for which patients must be carefully monitored. Conventional antipsychotic medications in low dosage may still have a role in a small proportion of patients, where there has been full remission and good tolerability; however, the indications are shrinking progressively. These principles are now accepted in most developed countries. (vi) Clozapine should be used early in the course, as soon as treatment resistance to at least two antipsychotics has been demonstrated. This usually means incomplete remission of positive symptomatology, but clozapine may also be considered where there are pervasive negative symptoms or significant or persistent suicidal risk is present. (v) Comprehensive psychosocial interventions should be routinely available to all patients and their families, and provided by appropriately trained mental health professionals with time to devote to the task. This includes family interventions, cognitive-behaviour therapy, vocational rehabilitation and other forms of therapy, especially for comorbid conditions, such as substance abuse, depression and anxiety. (vi) The social and cultural environment of people with schizophrenia is an essential arena for intervention. Adequate shelter, financial security, access to meaningful social roles and availability of social support are essential components of recovery and quality of life. (vii) Interventions should be carefully tailored to phase and stage of illness, and to gender and cultural background. (viii) Genuine involvement of consumers and relatives in service development and provision should be standard. (ix) Maintenance of good physical health and prevention and early treatment of serious medical illness has been seriously neglected in the management of schizophrenia, and results in premature death and widespread morbidity. Quality of medical care for people with schizophrenia should be equivalent to the general community standard. (x) General practitioners (GPs)s should always be closely involved in the care of people with schizophrenia. However, this should be truly shared care, and sole care by a GP with minimal or no special Optimal treatment of schizophrenia requires a multidisciplinary team approach with a consultant psychiatrist centrally involved.

Determination of fluoxetine and norfluoxetine enantiomers in human plasma by polypyrrole-coated capillary in-tube solid-phase microextraction coupled with liquid chromatography-fluorescence detection

A sensitive, selective, and reproducible in-tube polypyrrole-coated capillary (PPY) solid-phase microextraction and liquid chromatographic method for fluoxetine and norfluoxetine enantiomers analysis in plasma samples has been developed, validated, and further applied to the analysis of plasma samples from elderly patients undergoing therapy with antidepressants. Important factors in the optimization of in-tube SPME efficiency are discussed, including the sample draw/eject volume, draw/eject cycle number, draw/eject flow-rate, sample pH, and influence of plasma proteins. Separation of the analytes was achieved with a Chiralcel OD-R column and a mobile phase consisting of potassium hexafluorophosphate 7.5 mM and sodium phosphate 0.25 M solution, pH 3.0, and acetonitrile (75:25, v/v) in the isocratic mode, at a flow rate of 1.0 mL/min. Detection was carried out by fluorescence absorbance at Ex/Em 230/290 nm. The multifunctional porous surface structure of the PPY-coated film provided high precision and accuracy for enantiomers. Compared with other commercial capillaries, PPY-coated capillary showed better extraction efficiency for all the analytes. The quantification limits of the proposed method were 10 ng/mL for R- and S-fluoxetine, and 15 ng/mL for R- and S-norfluoxetine, with a coefficient of variation lower than 13%. The response of the method for enantiomers is linear over a dynamic range, from the limit of quantification to 700ng/mL, with correlation coefficients higher than 0.9940. The in-tube SPME/LC method can therefore be successfully used to analyze plasma samples from ageing patients undergoing therapy with fluoxetine. (C) 2009 Elsevier B.V. All rights reserved.

First detection of lead in black paper from intraoral film An environmental concern

Lead (Pb) contamination in the black paper that recovers intraoral films (BKP) has been investigated. BKP samples were collected from the Radiology Clinics of the Dental School of Ribeirao Preto, University of Sao Paulo, Brazil. For sake of comparison, four different methods were used. The results revealed the presence of high lead levels, well above the maximum limit allowed by the legislation. Pb contamination levels achieved after the following treatments: paper digestion in nitric acid, microwave treatment, DIN38414-54 method and TCLP method were 997 mu g g(-1), 189 mu g g(-1), 20.8 mu g g(-1), and 54.0 mu g g(-1), respectively. Flame atomic absorption spectrometry (FAAS) and inductively coupled plasma mass spectrometry (ICP-MS) were employed for lead determination according to the protocols of the applied methods. Lead contamination in used BKP was confirmed by scanning electron microscopy coupled with energy dispersive X-ray microanalysis (SEM-EDS). All the SEM imaging was carried out in the secondary electron mode (SE) and backscattered-electron mode (QBSD) following punctual X-ray fluorescence spectra. Soil contamination derived from this product revealed the urgent need of addressing this problem. These elevated Pb levels, show that a preliminary treatment of BKP is mandatory before it is disposed into the common trash. The high lead content of this material makes its direct dumping into the environment unwise. (C) 2009 Elsevier B.V. All rights reserved.

NEURAL CORRELATES OF SCENT MARKING BEHAVIOR IN C57BL/6J MICE: DETECTION AND RECOGNITION OF A SOCIAL STIMULUS

Mice show urinary scent marking behavior as a form of social communication. Marking to a conspecific stimulus mouse or odor varies with stimulus familiarity, indicating discrimination of novel and familiar animals. This study investigated Fos immunoreactivity in inbred C57BL/6J (C57) males following scent marking behavior in response to detection of a social stimulus, or discrimination between a familiar and an unfamiliar conspecific. In Experiment 1 C57 mice were exposed for four daily trials to an empty chamber; on a test day they were exposed to the same chamber or to a male CD-1 mouse in that chamber. Increased scent marking to the CD-1 mouse was associated with increased Fos-immunoreactive cells in the basolateral amygdala, medial amygdala, and dorsal and ventral premammillary nuclei. In Experiment 2 C57 mice were habituated to a CD-1 male for 4 consecutive days and, on the 5th day, exposed to the same CD-1 male, or to a novel CD-1 male. Mice exposed to a novel CD-1 displayed a significant increase in scent marking compared to their last exposure to the familiar stimulus, indicating discrimination of the novelty of this social stimulus. Marking to the novel stimulus was associated with enhanced activation of several telencephalic, as well as hypothalamic and midbrain, structures in which activation had not been seen in the detection paradigm (Experiment 1). These included medial prefrontal and piriform cortices, and lateral septum; the paraventricular nuclei, ventromedial nuclei, and lateral area of the hypothalamus, and the ventrolateral column of the periaqueductal gray. These data suggest that a circumscribed group of structures largely concerned with olfaction is involved in detection of a conspecific olfactory stimulus, whereas discrimination of a novel vs. a familiar conspecific stimulus engages a wider range of forebrain structures encompassing higher-order processes and potentially providing an interface between cognitions and emotions. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.

Molecular detection of Paracoccidioides brasiliensis in road-killed wild animals

Paracoccidioides brasiliensis infections have been little studied in wild and/or domestic animals, which may represent an important indicator of the presence of the pathogen in nature. Road-killed wild animals have been used for surveillance of vectors of zoonotic pathogens and may offer new opportunities for eco-epidemiological studies of paracoccidiodomycosis (PCM). The presence of P. brasiliensis infection was evaluated by Nested-PCR in tissue samples collected from 19 road-killed animals; 3 Cavia aperea (guinea pig), 5 Cerdocyon thous (crab-eating-fox), 1 Dasypus novemcinctus (nine-banded armadillo), 1 Dasypus septemcinctus (seven-banded armadillo), 2 Didelphis albiventris (white-eared opossum), 1 Eira barbara (tayra), 2 Gallictis vittata (grison), 2 Procyon cancrivorus (raccoon) and 2 Sphiggurus spinosus (porcupine). Specific P. brasiliensis amplicons were detected in (a) several organs of the two armadillos and one guinea pig, (b) the lung and liver of the porcupine, and (c) the lungs of raccoons and grisons. P. brasiliensis infection in wild animals from endemic areas might be more common than initially postulated. Molecular techniques can be used for detecting new hosts and mapping `hot spot` areas of PCM.

Genetic polymorphism in an inflammasome component, cervical mycoplasma detection and female infertility in women undergoing in vitro fertilization

The inflammasome is an inducible cytoplasmic structure that is responsible for production and release of biologically active interleukin-1 (IL-1). A polymorphism in the inflammasome component NALP3 has been associated with decreased IL-1 levels and increased occurrence of vaginal Candida infection. We hypothesized that this polymorphism-induced variation would influence susceptibility to infertility. DNA was obtained from 243 women who were undergoing in vitro fertilization (IVF) and tested for a length polymorphism in intron 2 of the gene coding for NALP3 (gene symbol CIAS1). At the conclusion of testing the findings were analyzed in relation to clinical parameters and IVF outcome. The frequency of the 12 unit repeat allele, associated with maximal inflammasome activity, was 62.3% in cases of female infertility vs. 75.6% in cases where only the male partner had a detectable fertility problem (p = 0.0095). Conversely, the frequency of the 7 unit repeat allele was 28.9% in those with a female fertility problem, 17.0% in women with infertile males and 18.4% in idiopathic infertility (p = 0.0124). Among the women who were cervical culture-positive for mycoplasma the frequency of the 7 unit repeat was 53.7% as opposed to 19.5% in those negative for this infection (p < 0.0001). We conclude that the CIAS1 7 unit repeat polymorphism increases the likelihood of mycoplasma infection-associated female infertility. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Cardiac MRI for Detection of Unrecognized Myocardial Infarction in Patients With End-Stage Renal Disease: Comparison With ECG and Scintigraphy

OBJECTIVE. The purposes of this study were to use the myocardial delayed enhancement technique of cardiac MRI to investigate the frequency of unrecognized myocardial infarction (MI) in patients with end-stage renal disease, to compare the findings with those of ECG and SPECT, and to examine factors that may influence the utility of these methods in the detection of MI. SUBJECTS AND METHODS. We prospectively performed cardiac MRI, ECG, and SPECT to detect unrecognized MI in 72 patients with end-stage renal disease at high risk of coronary artery disease but without a clinical history of MI. RESULTS. Fifty-six patients (78%) were men ( mean age, 56.2 +/- 9.4 years) and 16 (22%) were women ( mean age, 55.8 +/- 11.4). The mean left ventricular mass index was 103.4 +/- 27.3 g/m(2), and the mean ejection fraction was 60.6% +/- 15.5%. Myocardial delayed enhancement imaging depicted unrecognized MI in 18 patients (25%). ECG findings were abnormal in five patients (7%), and SPECT findings were abnormal in 19 patients (26%). ECG findings were false-negative in 14 cases and false-positive in one case. The accuracy, sensitivity, and specificity of ECG were 79.2%, 22.2%, and 98.1% (p = 0.002). SPECT findings were false-negative in six cases and false-positive in seven cases. The accuracy, sensitivity, and specificity of SPECT were 81.9%, 66.7%, and 87.0% ( not significant). During a period of 4.9-77.9 months, 19 cardiac deaths were documented, but no statistical significance was found in survival analysis. CONCLUSION. Cardiac MRI with myocardial delayed enhancement can depict unrecognized MI in patients with end-stage renal disease. ECG and SPECT had low sensitivity in detection of MI. Infarct size and left ventricular mass can influence the utility of these methods in the detection of MI.

Cancer detection and mammogram volume of radiologists in a population-based screening programme

This study investigates the relationship between the number of screening mammograms read by radiologists and the screening breast cancer detection rate. Cancer detection rates for incident screens (all women aged >= 40 years) were compared by increasing categories of reader volume using Poisson regression. Data from New South Wales (NSW) for a 2 year period (2000-2001) were obtained from the BreastScreen NSW programme. Cancer detection rates increased with the number of mammograms read in the programme, reaching a plateau of approximately 40 per 10,000 after 1375 mammograms per year. No significant differences in cancer detection were evident above 875 mammograms (compared to below 875 mammograms) per year (RR = 0.79, 95% CI 0.63-0.99). (c) 2005 Elsevier Ltd. All rights reserved.