Characterization of Thymic Nurse-Cell Lymphocytes,Using an Improved Procedure for Nurse-Cell Isolation

Thymic nurse cells (TNC), multicellular complexes consisting of lymphoid cells enclosed within cortical epithelial cells, were isolated from mouse thymus by a modified procedure allowing immunofluorescent labeling and flow cytometric analysis of their lymphoid contents (TNC-L). Collagenase was the only protease used for tissue digestion, to ensure that surface antigen markers remained intact. Zonal unit-gravity elutriation was used to enrich the TNC on the basis of their high sedimentation rate, followed by immunomagnetic bead depletion to remove residual mononuclear cell contaminants and a density separation to remove debris. The TNC-L were then released from inside TNC by a short period of culture. The measured contamination of TNC-L with exogenous thymocytes was around 0.5%. Three-color immunofluorescent labeling revealed that TNC-L included, as well as a maiority of immature CD4⁺8⁺3^low thymocytes, about 12% of apparently mature CD4⁺8^-3^high and CD4^-8⁺3^high thymocytes. TNC are located in the cortex, where mature cells are rare; the occurrence of mature phenotype cells within these structures suggests that they represent a microenvironment for the selection and generation of mature T cells.     

Depolarization-induced retrograde synaptic inhibition in the mouse cerebellar cortex is mediated by 2-arachidonoylglycerol

Endocannabinoids acting on CB₁ cannabinoid receptors are involved in short- and long-term depression of synaptic transmission. The aim of the present study was to determine which endocannabinoid, anandamide or 2-arachidonoylglycerol (2-AG), is involved in depolarization-induced suppression of inhibition (DSI) in the cerebellar cortex, which is the most widely studied form of short-term depression. Depolarization of Purkinje cells in the mouse cerebellum led to an increase in intracellular calcium concentration and to suppression of the inhibitory input to these neurons (i.e. DSI occurred). Orlistat and RHC80267, two blockers of sn -1-diacylglycerol lipase, the enzyme catalysing 2-AG formation, abolished DSI by acting downstream of calcium influx. In contrast, DSI occurred also in the presence of a phospholipase C inhibitor. Intact operation of the calcium-dependent messengers calmodulin and Ca²⁺–calmodulin-dependent protein kinase II were necessary for DSI. DSI was potentiated by an inhibitor of the main 2-AG-degrading enzyme, monoacylglycerol lipase. Interference with the anandamide metabolizing enzyme, fatty acid amide hydrolase, did not modify DSI. Thus, three kinds of observations identified 2-AG as the endocannabinoid involved in DSI in the mouse cerebellum: DSI was abolished by diacylglycerol lipase inhibitors; DSI was potentiated by a monoglyceride lipase inhibitor; and DSI was not changed by an inhibitor of fatty acid amide hydrolase. Further experiments indicated that 2-AG is the endocannabinoid mediating short-term retrograde signalling also at other synapses: orlistat abolished DSI in the rat cerebellum, DSI in the mouse substantia nigra pars reticulata and depolarization-induced suppression of excitation in the mouse cerebellum.     

Use of buccal cells collected in mouthwash as a source of DNA for clinical testing

CONTEXT: To maximize the participation rate in population genetic studies, alternatives to invasive whole blood collection are increasing. One such alternative is buccal epithelial cell collection, which, in contrast to venipuncture and finger sticks, is painless. Buccal cells, if collected and purified efficiently, offer an acceptable source for DNA to be used in research and clinical applications. OBJECTIVE: To develop a noninvasive sampling method for collecting cells for routine DNA testing in a clinical laboratory setting. DESIGN: Five factors were used to evaluate several brands of mouthwash: (1) compatibility with the DNA purification chemistry, (2) DNA yield, (3) DNA quality, (4) DNA stability at room temperature, and (5) mouthwash taste. Next, an optimization study was undertaken to maximize DNA yield. Finally, a validation study was undertaken with the optimized protocol to test a panel of 14 donors for DNA yield and performance and to test for the stability of DNA held in mouthwash. SETTING: Industrial research and development laboratory. RESULTS: Of 5 mouthwashes tested, Scope brand mouthwash received the highest overall ranking. The addition of proteinase K and glycogen to the protocol significantly enhanced DNA yields, with a test panel (n = 14) giving a range of 12 to 60 microg of DNA per donor. In a 4-week room temperature stability study, the DNA in mouthwash samples was found to be stable for at least 2 weeks. CONCLUSION: A clinically validated DNA purification chemistry was adapted to a noninvasive specimen collection method. This method used a commercially available mouthwash, Scope, to collect buccal epithelial cells for the preparation of high-quality DNA in high yield.     

The release of adenosine triphosphate from frog skeletal muscle in vitro

1. Active frog sartorius muscle in vitro liberates a substance into the bathing solution which has a pronounced stimulatory action on the frog heart.2. The stimulatory effect is not due to an increase in the K(+) concentration of the bathing solution, nor is it due to the liberation of catecholamines.3. In a molecular sieve chromatography procedure the stimulatory substance can be eluted in a single fraction which shows a maximum absorption of U.V. light at a wave-length of 265 nm, indicative of the presence of substances containing a purine ring.4. Low concentrations (10(-7)-10(-8) g/ml.) of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and uridine triphosphate (UTP) have a marked stimulatory action on the frog heart. The action of ATP and ADP on the heart is qualitatively very similar to that of the muscle bathing solution, while the action of UTP is distinctly different. The triphosphates of inosine, cytidine and guanosine stimulate the heart when in high concentration only. Adenosine and adenosine monophosphate do not stimulate the heart.5. Incubation of the muscle bathing solution and of solutions of ATP with the enzyme apyrase for the same time produces a similar marked reduction in the stimulatory action of both on the heart. Apyrase catalyses the break-down of nucleotide triphosphates to monophosphates.6. The elution behaviour of the stimulatory substance determined by molecular sieve chromatography is the same as that for ATP.7. The muscle bathing solution causes light to be emitted from firefly lantern extract, the pattern of light emission being similar to that produced by nucleotide triphosphates.8. The concentrations of ATP having the same quantitative action on the frog heart and on firefly extract as a given muscle bathing solution are almost identical, whereas the matching concentrations of ADP and UTP in the two methods of assay are widely different.9. It is concluded that ATP is released from active frog skeletal muscle in vitro. This release may play an important part in the reactive hyperaemia of muscular exercise since ATP has a powerful vasodilator action.     

Effects of methacholine and uridine 5'-triphosphate on tracheal mucus rheology in mice

We compared the action of methacholine (MCh) and uridine 5'-triphosphate (UTP) with and without pretreatment with the chloride channel blocker 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS) on the transepithelial potential difference (PD), the mucus collection rate (MCR), and tracheal mucus rheology using anesthetized C57BL/6 mice. The cystic fibrosis transmembrane conductance regulator (CFTR) blocker 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) was also used as a pretreatment for MCh. After collecting baseline mucus for 1.5 h, mucus secretion was stimulated by instilling 5 microl of 10(-2) M MCh or UTP around the upper trachea. There was a significant increase in PD after MCh or UTP stimulation (-21.3+/-2.0 mV MCh versus -14.1+/-1.6 mV control; -25.4+/-2.5 mV UTP versus -19.2+/-1.9 mV control). When UTP administration was preceded by DIDS, PD shifted from -15.2+/-2.9 to -12.0+/-2.2 mV. When MCh was preceded by DIDS or by NPPB, there was no change in PD. There was a significant decrease in mucus rigidity index, logG*, with MCh (2.54+/-0.09 versus 2.99+/-0.14 for control), similar to that previously reported in other species. With UTP, 14 of 16 mice responded in terms of PD becoming more negative, and of these, there was a significant difference in logG* after UTP administration (2.29 +/-0.10 versus 2.57+/-0.10 for control), whereas there was no change in logG* with DIDS administration before UTP. When DIDS administration preceded MCh, there was a diminished but still significant decrease in logG* from control, whereas there was no change in logG* when NPPB was preadministered. The control mucus collection rate was 0.19+/-0.09 mg/h, whereas after MCh stimulation, it increased to 2.83+/-0.78 mg/h. No significant difference was measured in the MCR after either UTP or DIDS+UTP stimulation. DIDS+MCh and NPPB+MCh both resulted in significant increases in MCR, but of a much smaller magnitude than that for MCh alone. We conclude that hypersecretion owing to UTP in C57BL/6 mice is less vigorous than with MCh, reflecting the limited population of Ca(2+)-dependent Cl(-) channels stimulated by UTP P(2) receptors. The action of MCh on tracheal mucus secretion in mice appears to involve both CFTR- and non-CFTR-dependent chloride channels.     

Knowledge of HIV infection and AIDS, and attitudes to testing and counselling among general practitioners in Northern Ireland.

All 922 general practitioners in Northern Ireland were sent a questionnaire on human immunodeficiency virus (HIV) infection and the acquired immune deficiency syndrome (AIDS). Five hundred and ninety four general practitioners (64.4%) returned the questionnaire. Thirty eight respondents (6.4%) knew of an HIV positive patient in their practice and 93.3% felt they should be informed if one of their patients was found to be HIV positive at a genitourinary medicine clinic, even without the patient's consent. Of the respondents, 76.8% were willing to be involved in the management of AIDS patients in their practice in cooperation with hospital colleagues but only 37.5% felt confident to provide AIDS counselling and advice. Of the 368 general practitioners who did not feel confident to provide AIDS counselling and advice, 41.3% felt that they had insufficient knowledge and 79.6% felt uncertain of their counselling skills. The information gathered on the administration of injections, taking blood samples and disposal of needles indicated that further education for general practitioners is required to ensure safety at work.     

Individual and cultural-diversity competency: focus on the therapist

The Competencies Conference: Future Directions in Education and Credentialing in Professional Psychology was held in Arizona in November 2002. One of the workshops, Individual and Cultural Differences (ICD), focused on racism, homophobia, and ageism. The consensus was that self-awareness and knowledge about the three "isms" are critical components in the education and training of psychologists. This article, authored by four of the workshop attendees, is a review of the current research and theoretical literature. Implications that address both content and context in graduate programs and training sites are presented. This is one of a series of articles published in this issue of the Journal of Clinical Psychology. Several other articles that resulted from the Competencies Conference will appear in Professional Psychology: Research and Practice and The Counseling Psychologist.     

Control of dynamic and static nuclear bag fibres and nuclear chain fibres by gamma and beta axons in isolated cat muscle spindels.

1. The behaviour of nuclear bag and nuclear chain intrafusal fibres in isolated cat muscle spindles with a blood supply, during stimulation of dynamic gamma axons, dynamic beta axons, or static gamma axons in ventral root filaments was observed and recorded on still and moving film. 2. Most spindles were controlled by one dynamic gamma axon (sometimes a beta axon) and three static gamma axons, one of which was often non-selective in distribution. A large majority of fusimotor axons controlled one pole of the spindle only. 3. Dynamic gamma and beta axons produced focal contraction in only one of the two nuclear bag fibres in any spindle and this fibre was never activated by static gamma axons. Maximal tetanic contraction was attained slowly and the primary sensory spiral on this fibre was stretched by a small amount only. This fibre has been named the 'dynamic nuclear bag fibre'. 4. Static gamma axons produced either: (a) focal contraction in the second of the two nuclear bag fibres only; (b) local contraction in the bundle of nuclear chain fibres only; or (c) contraction in one nuclear bag fibre and the nuclear chain fibres together. Maximum tetanic contraction of this nuclear bag fibre stretched its primary sensory spiral considerably and the time to plateau was relatively short. This fibre has been named the 'static nuclear bag fibre'. 5. 'Driving' of the Ia afferent discharge could always be produced by non-selective static gamma axons, frequently by static gamma axons controlling nuclear chain fibres alone, and was probably due to mechanical oscillation in nuclear chain fibres. It was never produced by dynamic gamma axons and on one occasion only by a static gamma axon controlling a nuclear bag fibre alone. 6. The conduction velocities of dynamic gamma and static gamma axons overlapped extensively, though dynamic gamma axons were absent from the lower end, and static gamma axons innervating nuclear chain fibres only were absent from the upper end, of the range of velocities. 7. The observations are correlated with spindle structure and histochemistry. Dynamic and static nuclear bag fibres are shown to correspond with 'bag1 fibres' and 'bag2 fibres', respectively (Ovalle & Smith, 1972). 8. The possible origin of the dynamic and static actions of fusimotor axons and the role of the dynamic and static intrafusal systems in motor control are discussed.     

Antibody response to dengue-2 vaccine measured by two different radioimmunoassay methods

Two different radioimmunoassays were used to detect virus-specific antibodies in sera from human volunteers inoculated with an attenuated dengue type 2 (DEN-2) vaccine (PR-159/S-1). An indirect radioimmunoassay required purified DEN-2 virions for optimal reactivity but was 10 to 500 times more sensitive than neutralization or hemagglutination inhibition tests. An antibody capture radioimmunoassay was able to utilize crude antigens from either DEN-infected mouse brains or Aedes albopictus cell culture supernatants. When the two radioimmunoassay techniques were compared, the indirect method appeared to be the best assay for immunoglobulin G (IgG), whereas the antibody capture method was more sensitive for IgM detection. Selected human sera were examined for IgG, IgM, and IgA responses by using both techniques at various intervals after immunization. Although there were differences in magnitude, yellow fever immune as well as flavivirus nonimmune volunteers responded to DEN-2 vaccination by demonstrating IgG, IgM, and IgA antibody responses. In the nonimmune group, the most prevalent immunoglobulin detected was IgM, whereas in the yellow fever immune group, the predominant post-DEN-2 vaccine immunoglobulin was IgG. The preponderance of DEN-2-specific neutralizing antibodies were associated with either IgM or IgG according to the immune status of the volunteer. All classes of immunoglobulins attained maximum levels between 21 and 60 days postvaccination. In the majority of volunteers, IgM responses were relatively transient and could not be detected 6 months after immunization, whereas IgG and IgA antibodies were still detectable after this period.     

Skin testing and extrinsic allergic alveolitis

Skin testing with six common allergens, tuberculin and a sterile avian antigen preparation from pigeon serum was performed on 102 pigeon fanciers. The incidence of positive prick tests to common allergens was no different for subjects with extrinsic allergic alveolitis. EAA, caused by avian exposure than the whole group. Positive immediate weal and flare reactions following skin prick testing with avian antigen occurred in 22 subjects and was closely correlated with atopy. However, when the same antigen was administered intradermally, 69 subjects developed an immediate (15 min) weal and flare reaction which did not correlate with atopy, instead, the weal diameter correlated significantly with the serum IgG antibody titre against pigeon serum gamma-globulin antigen, and furthermore, the higher grades of reaction were highly selective for subjects with EAA. Ten subjects, all with strong early intradermal skin reactions, developed a late (4-6 h) skin reaction; this was again highly selective for EAA. The subjects with cutaneous anergy to tuberculin had markedly higher IgG antibody titres to avian antigens, and these included the majority of the subjects with alveolitis.