Flow cytometric quantitation of yeast a novel technique for use in animal model work and in vitro immunologic assays

Animal models of fungal and other infectious diseases often require that the number of organisms in tissue be quantified, traditionally by grinding organs, plating them on agar and counting colony forming units (CFU). This method is labor intensive, slow as some fungi require two weeks of culture and limited in reliability by poor plating efficiency. To circumvent these problems, we developed a flow cytometric method to quantify yeast. In vitro cultured Blastomyces dermatitidis, Cryptococcus neoformans, Candida albicans and Histoplasma capsulatum yeast were labelled with specific monoclonal or polyclonal antibodies to stain surface determinants or with Calcofluor to stain cell-wall chitin. A defined number of fluorescently labelled beads were added prior to acquisition by flow cytometry as a reference standard for quantitation. Beads were readily distinguished from yeast by forward scatter, side scatter and intensity of fluorescence. Cultured yeast were enumerated by both standard CFU determination and flow cytometry in a range of 10(2) to 10(7) cells. Only flow cytometry enabled discrimination of live and dead yeast by using appropriate fluorescent dyes. The flow cytometric method was applied to murine models of histoplasmosis and blastomycosis to quantify the burden of fungi in the lungs of infected mice. Labelling yeast with Calcofluor alone resulted in unacceptably high levels of nonspecific binding to mouse cell debris. In contrast, labelling H. capsulatum with a rabbit polyclonal antiserum and B. dermatitidis with a monoclonal antibody to the surface protein WI-1 permitted accurate quantitation. We conclude that this flow cytometry technique is rapid, efficient and reliable for quantifying the burden of infection in animal models of fungal disease. The technique also should lend itself to performing cytotoxicity assays that require discrimination of live and dead fungi, or phagocytosis assays that require discrimination of intracellular and extracellular organisms.     

Dexamethasone concentration in vitreous and serum after oral administration

OBJECTIVE: To assess the outcomes of changes in mental health policy introduced in Italy in 1978. METHODS: Data on psychiatric services, before and after the policy change, are presented. Effects of change are evaluated through indicators related to four issues: transfer of care, criminalisation of the mentally ill, suicides, and homelessness. RESULTS: Admissions of new patients to mental hospitals have been stopped and the size of the mental hospital population is now very low (26 per 100,000 population). Psychiatric care has been shifted to community services including general hospital psychiatric units. There has been an overall reduction of psychiatric hospitalisation. However, the provision of residential facilities is inadequate and community services are unevenly distributed across the country. Few negative effects of changing patterns of care have been reported, although the low quality of data limits the validity of such a conclusion. Outcome of care in areas where the full range of community services is available has been rated as satisfactory. CONCLUSIONS: Although care of the mentally ill has been shifted to community services, we lack hard data on the social and clinical outcome of community care at the nation-wide level. Long-term monitoring and evaluation of community services is a high priority in Italy.     

Parenting children with anorectal malformations: implications and experiences

Parents play a crucial role in the life of a child suffering from an anorectal malformation (ARM), since their guidance contributes to the degree to which the child learns to cope with his or her disability. We investigated whether they experience stress in parenting such a child and also attempted to identify somatic or behavioral characteristics in the child that influence the stress of parenting. The parents of 109 children (69 males, 40 females; median age 5.9 years, range 1-18 years) with an ARM (58 low, 10 intermediate, 41 high) were studied. The Nijmegen Questionnaire on Child-rearing Situations (NQCS) was used to investigate the existing parenting situation. Behavioral characteristics of the children were studied by means of the Child Behaviour Checklist (CBCL) and the Teacher Report Form (TRF). In a semi-structured interview, we investigated how parents experienced the implications of the disability in everyday life with their child. Our study showed that as far as the perception of parenting stress is concerned, parents of children with an ARM do not differ from those with healthy primary-school children. Within the group of parents with ARM-afflicted children, the parents of older, incontinent children experienced relatively more stress, especially when the child concerned was male. With regard to the children's behavior, the parents and teachers under investigation did not report a higher than normal incidence of deviant behavior. However, when individual parents observed difficult behavior in their child, they found it harder to deal with than the incontinence for feces. Regarding the implications of the disorder for their everyday lives, parents were concerned and indicated a need for specific counselling. We conclude that having a child with a somatic affliction, in this case an ARM, does not automatically imply that the parents experience child-rearing problems. However, certain groups of parents are more at risk, i.e., parents with older, incontinent sons and parents with children exhibiting behavioral problems. In addition, our study shows that parents do have difficulties in coping with the implications of the disorder and express a need for support. We feel that patient care can be improved if aid is tailored to these specific problems.     

Is nondipping in 24 h ambulatory blood pressure related to cognitive dysfunction?

OBJECTIVE: Associations between the outcome of 24 h ambulatory monitoring and cognitive performance were studied in order to evaluate the potential relevance of ambulant blood pressure status to brain function. It was hypothesized that a small daytime-night-time difference in mean blood pressure (nondipping) is associated with reduced cognitive performance, in line with studies in hypertensive subjects that have reported associations between nondipping and target-organ damage. METHODS: The study followed a cross-sectional design and was part of a larger research programme on determinants of cognitive aging (Maastricht Aging Study, MAAS). A group of 115 community residents aged 28-82 years was recruited from a general practice population and screened for cardiovascular events and medication use. All underwent 24 h blood pressure monitoring. Cognitive performance was measured with tests of verbal memory, attention, simple speed and information processing speed. RESULTS: Mean daytime or night-time levels of both systolic and diastolic blood pressure were unrelated to cognitive outcome, when age, sex and educational level were controlled for. Differences between mean daytime and night-time blood pressure (based on both narrow and wide measurement intervals for day and night-time periods) were positively associated with memory function (5-9% of additional variance explained) and one sporadic positive association was found on the sensorimotor speed score (4%). Nondippers (n=15) showed lower levels of both memory and sensorimotor speed scores. CONCLUSIONS: Ambulatory blood pressure status was not associated with cognitive performance. A reduced nocturnal blood pressure drop was associated with quite specific cognitive deficits, but the underlying mechanism remains to be determined.     

Convulsions due to increased permeability of the blood-brain barrier in experimental cerebral malaria can be prevented by splenectomy or anti-T cell treatment

Experimental cerebral malaria (ECM) can be induced in C57B1 mice by infection with Plasmodium berghei K173 parasites. Behavioral changes shortly before they die of ECM may reflect disturbance of the integrity of the blood-brain barrier (BBB). Folic acid elicits strong convulsive activity if the permeability of the BBB is increased. Administration of folic acid to mice during development of ECM induced convulsions. Interventions known to prevent fatal outcome from ECM, such as splenectomy or treatment with anti-CD4 or anti-CD8 monoclonal antibodies, also prevented sensitivity to folic acid-induced convulsions. In addition, infected mice with ECM and sensitive to folic acid-induced convulsions, recovered from this sensitivity after treatment with anti-T cell antibodies within 4 h. These data suggest that disturbance of the permeability of the BBB can be reversed and depends on the involvement of T cells.     

A powerful GABA(B) receptor-mediated inhibition of GABAergic neurons in the arcuate nucleus

We combined histofluorescence with in situ hybridization to identify GABAergic neurons in the arcuate nucleus (ARC) following electrophysiological recordings, using GAD65 as a marker. Intracellular recordings were made in hypothalamic slices prepared from ovariectomized guinea pigs. Over 90% of ARC neurons tested with the GABA(B) receptor agonist baclofen responded with a membrane hyperpolarization or an outward current. The hyperpolarization was dose-dependent, and the GABA(B) receptor antagonist CGP 35,348 produced a rightward shift in the agonist dose-response curve. Agonist potency was lower, and the efficacy greater, in GAD-positive neurons. The use of this novel technique for identifying GABAergic neurons thus reveals differences in the pharmacodynamics of GABA(B) receptor activation GABAergic and non-GABAergic ARC neurons.     

Cognitive ability and cerebral lateralisation in transsexuals

INTRODUCTION: The application of high-frequency current to the AV junctional area results in a temperature rise in the myocardium and may cause accelerated junctional rhythm (AJR). The aim of the study was to characterize heat-induced AJR in an in vitro animal model. METHODS AND RESULTS: Studies were performed in isolated perfused pig and rabbit hearts. Using a small heating probe, we could induce AJR from a discrete area located in the middle of the triangle of Koch, which was smaller than the area from which RF energy application could elicit AJR. Histology showed that the heat-sensitive area was located over, or close to, the compact AV node. It did not correspond with the areas where double potentials were found or with the site(s) of earliest atrial activation during VA conduction. Microelectrode recordings revealed that AJR arose in nodal-type cells. Heat increased the slope of the phase 4 depolarization and shortened the action potential duration. Two types of AJR were observed: the first one was regular and the second one showed irregularity in the intervals. Interaction of multiple foci and the presence of conduction block between the foci and the His bundle caused the irregularity of the His-His intervals during the second type of AJR. CONCLUSION: AJR observed during heat and RF application in the AV nodal area results from the effect of heat on AV nodal cells with underlying pacemaker activity. The heat-sensitive area is located over, or very close to, the compact AV node.     

Definition of natural T cell antigens with mimicry epitopes obtained from dedicated synthetic peptide libraries

Progress has recently been made in the use of synthetic peptide libraries for the identification of T cell-stimulating ligands. T cell epitopes identified from synthetic libraries are mimics of natural epitopes. Here we show how the mimicry epitopes obtained from synthetic peptide libraries enable unambiguous identification of natural T cell Ags. Synthetic peptide libraries were screened with Mycobacterium tuberculosis-reactive and -autoreactive T cell clones. In two cases, database homology searches with mimicry epitopes isolated from a dedicated synthetic peptide library allowed immediate identification of the natural antigenic protein. In two other cases, an amino acid pattern that reflected the epitope requirements of the T cell was determined by substitution and omission mixture analysis. Subsequently, the natural Ag was identified from databases using this refined pattern. This approach opens new perspectives for rapid and reliable Ag definition, representing a feasible alternative to the biochemical and genetic approaches described thus far.     

The use of oral contraceptives by adolescents for contraception, menstrual cycle problems or acne

BACKGROUND: Oral contraceptives are prescribed as contraception but also as therapy for menstrual cycle disturbances and acne. We studied the prevalence of oral contraceptive (OC) use and the indications to start OC use among adolescents. METHODS: A cohort consisting of ninth grade secondary school girls (mean age 15.3+/-0.6 (s.d.) years) answered a questionnaire on their menstrual cycle. OC users were asked about duration and reasons for OC-use and the name of the preparation they used. The influence of calendar age, gynecological age and level of education on the prevalence of OC was studied by multiple logistic regression analysis. RESULTS: The response on the questionnaire was 92%. Of 2248 responders 248 (11%) used oral contraceptives: 74% used low dose 'sub 50' preparations, 3% pills with 50 microg estrogen, 3% tri-phase preparations and 17% pills with antiandrogens. Of girls aged 14, 15 and 16 years 4%, 12% and 28% respectively, used OC. Of the 15-year-olds 31% mentioned contraception as most important reason for OC use, 18% menstrual cycle irregularity, 26% dysmenorrhea, 10% acne and 5% other reasons. Calendar age, gynecological age and level of education were independent variables for OC use in general and for OC use for contraception or dysmenorrhea, but less so for OC use for menstrual cycle irregularity or acne. CONCLUSIONS: During adolescence low dose OC's were frequently used. In The Netherlands OC use among girls aged 15 and 16 years doubled in comparison with 1982. One third of the adolescent OC-users mentioned contraception as most important reason to start OC. Gynecological age (a determinant of biological maturation), calendar age (a determinant of biological maturation and lifestyle in peer groups), and level of education (a determinant of lifestyle in peer groups) were associated with OC use.