Intracellular Staining Techniques Research Articles

Effect of Cannabidiol on Human Peripheral Blood Mononuclear Cells and CD4+ T Cells.

Cannabidiol (CBD), the main non-psychoactive component of Cannabis sativa L., is widely used in therapy for the treatment of different diseases and as an adjuvant drug. Our aim was to assess the effects of CBD on proinflammatory cytokine production and cell proliferation in human peripheral blood mononuclear cells (PBMCs) and on CD4+ T lymphocyte differentiation, and, furthermore, to test CBD's ability to affect the functional properties of regulatory T cells (Treg). Experiments were performed on isolated PBMCs and purified CD4+ T lymphocytes obtained from the buffy coats of healthy subjects. Cytokines produced by CD4+ T cells were evaluated by flow cytometry and intracellular cytokine staining techniques. PBMC cytokine production was measured by an ELISA assay. Real-time PCR was used to assess the mRNA expression of cytokines and the key transcription factors (TFs) of CD4+ T cells. Finally, the proliferation of PBMC and CD4+ T effector cells (Teff), alone and in the presence of Treg, was assessed by flow cytometry. Results showed that CBD affects both the frequency of IL-4-producing CD4+ and of IFN-γ/IL-17-producing cells and dramatically decreases the mRNA levels of all TFs. Stimuli-induced cytokine mRNA expression was decreased while protein production was unaffected. CBD was unable to affect the ability of Treg to prevent Teff cell proliferation while it slightly increased PBMC proliferation. In conclusion, CBD may inhibit the expression of proinflammatory cytokines; however, the effect of CBD on cell proliferation suggests that this cannabinoid exerts a complex activity on human PBMCs and CD4+ T cells which deserves further investigation.

Identification of circulating regulatory T lymphocytes with membrane markers - a new multiparameter flow cytometry protocol.

Regulatory T cells (Tregs) are a unique CD4+ T cell subset involved in the regulation of immune responses. The traditional immunophenotype used to define Tregs includes CD4+CD25high and the expression of the transcription factor Forkhead box protein 3 (FoxP3). A complex technique of intracellular staining, transient upregulation of FoxP3 in activated conventional T lymphocytes (Tcons), and the omission of naïve CD45RA+ Tregs with downregulated FoxP3 activity but a demethylated FOXP3 promoter region may lead to inaccurate quantification. In an attempt to meet the need for a reliable and simplified enumeration strategy, we investigated different membrane markers to capture the entire Treg compartment and to identify subpopulations of Tregs. Analyses were performed on whole blood. Tested gating strategies were based on the expression of the following membrane antigens: CD45, CD3, CD4, CD25, CD127, CD26, CD6, CD39, CD71, HLA-DR, CD45RA and CD31. Double controls with FoxP3 were performed. The final enumeration panel consisted of the membrane markers CD45, CD3, CD4, CD25, CD127, CD26, CD39, CD45RA and CD31. A deep analysis of T cells with the CD4+CD25+CD127low/-CD26low/-CD45RAimmunophenotype revealed high expression of FoxP3 and/or CD39, while cells with the naïve immunophenotype, CD4+CD25+CD127low/-CD26low/-CD45RA+, presented lower expression of suppressor markers. Antigen CD31 is considered to be a valuable membrane marker of thymus-derived Tregs. The presented 9-color panel that can be easily applied in laboratories enables reliable enumeration of Tregs with additional information about the functionality, maturity and origin of T regulatory cells.

Upregulation of CD4+ T-Lymphocytes by Isomeric Mixture of Quercetin-3-O-Rutinoside and Quercetin-3-O-Robinobioside Isolated from Millettia aboensis

ABSTRACTBackground: Millettia aboensis (Hook. F.) Baker (Fabaceae) is popular in ethnomedicine for its acclaimed efficacy in a number of disease conditions. This study evaluated the immunomodulatory effect of the leaf extract as a possible mechanism of its ethnomedicinal uses.Methods: Humoral and cellular immune responses of Balb/c mice to tetanus toxoid and cyclophosphamide, respectively, were used to monitor immunomodulatory activities of the ethanol leaf extract and fractions of M. aboensis at 200, 300 and 400 mg/kg. Active (butanol) fraction of the extract was subjected to chromatographic purifications to isolate the active compound and the structure elucidated by a combination of 1D and 2D NMR and mass spectrometry. Stimulation of specific T-lymphocytes using intracellular cytokine staining technique was used to evaluate immune-enhancing activity of the isolated compound.Results: The extract and fractions evoked increase in both humoral and cellular immunity. At 400 mg/kg of butanol fraction, the normalized mean secondary production of IgG1 and IgG2a antibodies were 9.0 and 7.7, respectively. Serum cytokine production by butanol fraction following secondary challenge with tetanus toxoid showed that IL-12, IL-17A and IFN-γ were expressed by 48.14, 41.37 and 38.22%, respectively. Structural elucidation of the active compound revealed presence of isomeric mixtures of quercetin-3-O-rutinoside and quercetin-3-O-robinobioside (Compound 1a/b). Compound 1a/b exhibited in vitro upregulation of specific CD4+ T-lymphocytes that were largely IFNγ releasing with up to 43.7% stimulation at 6.25 μg/mL compared to the baseline effect in DMSO vehicle control group.Conclusion: M. aboensis expressed strong immune-enhancing properties, which may explain its ethnopharmacological use in disease management.

Flow Cytometric Analysis of Protective T-Cell Response Against Pulmonary Coccidioides Infection.

The incidence of systemic fungal infections has increased throughout the world, spurring much interest in developing effective vaccines. Coccidioidomycosis, also known as San Joaquin Valley fever, is a potentially life-threatening respiratory mycosis. A vaccine against Coccidioides infection would contribute significantly to the well-being of the approx. 30 million residents in the Southwestern USA as well as the multitude of travelers who annually visit the endemic regions. We have applied a live, attenuated vaccine (∆T) to explore the nature of vaccine immunity in mice after intranasal challenge with a potentially lethal dose of Coccidioides spores. Coccidioides spores are airborne and highly infectious for mammalian hosts and classified as a biosafety level 3 agent. T cells are critical in the development of protective immunity against a variety of microorganisms as well as the development of autoimmune disease and allergic responses. Profiles of cytokines detected in lung homogenates of ∆T-vaccinated mice were indicative of a mixed Th1, Th2, and Th17 immune response. We have developed an intracellular cytokine staining and flow cytometric (ICS) technique to measure activated CD4(+) and CD8(+) T cells and IFN-γ-, IL-4-, IL-5-, and IL-17A-producing T cells in the lungs of mice that are challenged with a potentially lethal dose of Coccidioides spores. The numbers of pulmonary Th1 and Th17 cells during the first 2 weeks post-challenge showed a progressive increase in vaccinated mice and corresponded with reduction of fungal burden. In this protocol, we describe the methodology for culture and isolation of the live, attenuated ΔT spores of Coccidioides used to vaccinate mice, preparation of pulmonary cells, and staining protocol for cell surface markers and intracellular cytokines. This is the most reliable and robust procedure to measure frequencies and numbers of each selected T-cell subsets in lungs of vaccinated versus control mice and can be readily applied to evaluate T-cell response against other microbial infections.

Distribution and density of contacts from noradrenergic and serotonergic boutons on the dendrites of neck flexor motoneurons in the adult cat.

Serotonergic (5-HT) and noradrenergic (NA) input to spinal motoneurons is essential for generating plateau potentials and self-sustained discharges. Extensor motoneurons are densely innervated by 5-HT and NA synapses and have robust plateau potentials and self-sustained discharges. Conversely, plateau potentials and self-sustained discharges are very rare in flexor motoneurons. The most likely reasons for this difference are that flexor motoneurons have few 5-HT and NA synapses and/or they are distributed distant to the channels responsible for plateau potentials and self-sustained discharges. However, the distribution of 5-HT and NA synapses on flexor motoneurons is unknown. Here we describe the distribution and density of 5-HT and NA synapses on motoneurons that innervate the flexor neck muscle, rectus capitis anterior (RCA), in the adult cat. Using a combination of intracellular staining, fluorescent immunohistochemistry, and 3D reconstruction techniques, we found that 5-HT and NA synapses are widely distributed throughout the dendritic trees of RCA motoneurons, albeit with a strong bias to small-diameter dendrites and to medial dendrites in the case of NA contacts. The number of 5-HT and NA contacts per motoneuron ranged, respectively, from 381 to 1,430 and from 642 to 1,382, which is 2.3- and 1.4-fold less than neck extensor motoneurons (Montague et al., J Comp Neurol 2013;521:638-656). These results suggest that 5-HT and NA synapses on flexor motoneurons may provide a powerful means of amplifying synaptic currents without incurring plateau potentials or self-sustained discharges. This feature is well suited to meet the biomechanical demands imposed on flexor muscles during different motor tasks.

P0045 Ubiquitinated proteins collected by a ubiquitin binding adaptor protein VX3 as a novel cancer vaccine

<h3>Background</h3> Our previous studies have shown that autophagosome-enriched vaccine named Dribble sequesters both long-lived and short-lived proteins, as well as damage-associated molecular pattern (DAMP) molecules; we also found that these Dribbles are efficient antigen carriers. Moreover, other studies showed that the concentration of ubiquitinated proteins (Ub-Ps) is increased in many cancer types. In this study, we tried to use the Ub-Ps that were isolated from the lymphoma cell line (EG7.OVA) as a novel cancer vaccine. <h3>Methods</h3> We isolated the Ub-Ps from the whole cell lysate of EG7.OVA cells using two ubiquitin binding proteins (TUBEs and VX3-eGFP). Isolated Ub-Ps were tested by SDS PAGE and Western blot. Four C57BL/6 mice groups were vaccinated by Ub-enriched proteins, Ub-depleted proteins, whole cell lysate, and PBS respectively. Three groups of C57BL/6 mice (three mice per group) have been vaccinated with 30μg, 100μg, and 300μg, respectively, of Ub-enriched proteins. Lymphocytes from each group were co-cultured with inactivated EG7.OVA cells. Secreted IFN-<i>γ</i> concentration was detected by ELISA and an intracellular staining technique. Lymphoma model was established using EG7.OVA cells. Tumour growth was monitored. <h3>Findings</h3> We found that the level of IFN-<i>γ</i> in the Ub-Ps vaccinated group was significantly higher than in the other three groups. We also found that the level of IFN-<i>γ</i> is increased by increasing the injected Ub-Ps dose. Ub-Ps vaccine showed a significant inhibitory effect on tumour growth compared with the other three groups. <h3>Interpretation</h3> We conclude that this vaccine is dose-dependent. These primary results give us a hope that the Ub-Ps could be used as a potent anti-cancer vaccine.

Issue Highlights—May 2013

Issue Highlights—May 2013

Quantification of pluripotency transcription factor levels in embryonic stem cells by flow cytometry.

Embryonic stem (ES) cell lines are derived from the inner cell mass of the pre-implantation blastocyst and are characterized by the ability to undergo indefinite self-renewal while retaining the potential to differentiate into each of the three primary germ layers. The ability of individual ES cells to self-renew or appropriately respond to differentiation signals is influenced by the intracellular level of a number of crucial transcription factors. It is therefore important to be able to reliably quantify the levels of these proteins in single cells. Here we present an intracellular staining technique for flow cytometry suitable for monitoring transcription factor expression in ES cells. We illustrate the application of this technique to the detection of Oct4 and Nanog proteins and the coupling of this approach with fluorescent reporters of gene activity.

Alteration of CD8+ T cell effector diversity during HIV‐1 infection with discordant normalization in effective antiretroviral therapy

Although the impairment of HIV-specific T lymphocytes is evident during chronic HIV-infection, it is unclear whether the increased CD8+ T cells associates with either selective or overall change of effector functional phenotype. Instead of study on HIV-specific T cells only, analyzing bulk T cell populations represent a neglected area of T cell impairment, which go far beyond HIV-specific T cells. In this study, we determined the diversity of CD8+ T cells in term of cytolytic molecule expression (perforin, granzyme A, and granzyme B) and cytokine production ability (IFN-gamma, TNF-alpha, and IL-2) using intracellular staining and flow cytometry technique. The results were compared between healthy individuals, untreated, and antiretroviral therapy (ART) treated HIV infected patients. We demonstrated the presence of four different subsets of CD8+ T cells that expressed different combinations of cytolytic molecules. We also identified seven different subsets of cytokine producing cells based on different combination of IFN-gamma, TNF-alpha, and IL-2. Results showed significant alterations of these cell subsets that expressed different combination of cytolytic effector molecules or cytokines in HIV infected patients. Furthermore, cytolytic molecule expressing cell subsets are not normalized in effective ART treated patients, whereas the selective population of cytokine producing cells returned to normal value. The effector diversity of CD8+ T cells changed in HIV infected patients. Although effective ART altered functional diversity of these cells, long-term suppression of viral replication may be required to normalize the selected CD8+ T cell effector phenotype in HIV infected patients.

Offset response of the olfactory projection neurons in the moth antennal lobe

We investigated a population activity of central olfactory neurons after the termination of odor input. Olfactory response of projection neurons in the moth primary olfactory center was characterized using in vivo intracellular recording and staining techniques. The population activity changed rapidly to the different states after the stimulus offset. The response after stimulus offset represents information regarding odor identity. We analyzed the spatial distribution of offset-activated glomeruli in a virtual neuronal population that was reconstructed using accumulated individual recordings obtained from different specimens. The offset-activated glomeruli tended to be widely distributed, whereas the onset-activated glomeruli were relatively clustered. These results suggest the importance of lateral interaction in shaping the offset olfactory response.

Tregs and Human Atherothrombotic Diseases

Immunology has recently penetrated the field of atherothrombosis. A number of human and experimental studies have documented that both cellular and molecular immune effectors are involved at various stages of the pathological process.1 This has directed our attention toward the use of immunomodulatory strategies in atherothrombosis; these strategies are commonly used for the treatment of other chronic inflammatory diseases. Notably, the administration of immunosuppressive drugs is the most common approach for treating immune-mediated diseases. However, the long-term administration of non–antigen-specific agents that cannot distinguish between beneficial and destructive immune responses is a major drawback of this strategy. See accompanying articles on pages 1825 and 1832 Interesting alternatives consist of strategies aimed at potentiating the physiological regulatory mechanisms, if defective. In the field of atherothrombosis research, experimental studies have clearly shown that a defect in regulatory CD4+ T cells (Tregs) favors atherogenesis.2 Based on their potent immunosuppressive activity, the expansion of the Treg compartment by cell transfer or the administration of stimulating molecules can be envisaged as a new therapeutic strategy. These findings have provided a strong incentive to perform clinical studies with the aim of assessing whether a defective Treg compartment is associated with the progression of atherosclerotic disease and the occurrence of atherothrombotic manifestations in humans. Three clinical studies,3–5 in a limited number of patients with coronary artery disease, first reported that patients with acute coronary syndromes have fewer and/or inefficient Tregs in their blood. In the present issue of Arteriosclerosis, Thrombosis, and Vascular Biology , a larger clinical study6 reports interesting challenges to these findings; a second study7 explores the role of Tregs in stenotic and dilative pathological remodeling …

Arrangement of Output Information from the 3 Macroglomerular Units in the Heliothine Moth Helicoverpa assulta: Morphological and Physiological Features of Male-Specific Projection Neurons

Helicoverpa assulta is exceptional among heliothine species studied so far as concerns composition of the pheromone blend. Previous reports have accordingly pointed out distinct characteristics in the male-specific olfactory pathway of this species, peripherally by an unusual distribution of 2 sensillum categories and centrally by a particular anatomical arrangement of the male-specific glomeruli constituting the macroglomerular complex (MGC). In order to determine the physiological tuning of the 3 MGC units in this species, we have characterized male-specific antennal-lobe projection neurons morphologically and physiologically by use of the intracellular recording and staining technique combined with confocal microscopy. The results show 2 projection neuron types of equal numbers, one that responds to the primary pheromone component, cis-9-hexadecenal, and arborizes in the cumulus and one that responds to the interspecific signal, cis-9-tetradecenal, and arborizes in the dorsomedial unit. A third type responded to the secondary pheromone component, cis-11-hexadecenal, and innervated the smaller ventral unit. The results complement previous findings from tracing of physiologically identified receptor neurons and determine for the first time the functional specificity of each glomerulus in the MGC of H. assulta. The results are particularly interesting because heliothine moths are attractive objects for comparative studies addressing questions concerning divergence of male-specific olfactory characteristics related to speciation.

Distribution of Dendrites from Longissimus Lumborum Motoneurons Stained Intracellularly with Biocytin in Adult Cats

The three-dimensional distribution of dendrites from motoneurons innervating longissimus lumborum (Long Motoneurons) in the L4 spinal segment was examined in the adult cat using intracellular staining techniques. Long Motoneurons were electrophysiologically identified, stained with injection of biocytin and reconstructed from serial histological sections. Somas of Long Motoneurons were mainly located in the lateral-ventral area of the ventral horn. The dendritic distribution followed an orderly pattern in all motoneurons examined. Long Motoneurons showed a multi-directional distribution of dendrites, and the dendritic distribution pattern varied depending on the motoneuron. All studied motoneurons distributed dendrites from the spine into the white matter. The most significant morphological characteristic of the Long Motoneurons was the variation in their dendritic distribution. No relationship was observed between the effects of peripheral afferent inputs from the hindlimb and morphological characteristics of motoneurons.

Flow cytometric measurements of TB‐specific T cells comparing with QuantiFERON‐TB gold

Interferon-gamma (IFN-gamma) release assays and the detection of IFN-gamma synthesis in the cytoplasm of activated CD4+ T cells by flow cytometry have recently been used for tuberculosis (TB) diagnosis. The aim of this study was to compare the performance of IFN-gamma assay between ELISA (QuantiFERON-TB Gold, QFT) and intracellular cytokine flow cytometry (ICCFC), and to investigate the significance of an optimal gating strategy in ICCFC. The CD4+ T cell response to TB antigens was measured using the intracellular cytokine staining technique and four color FC (CD3, CD4, IFN-gamma, and tumor necrosis factor-alpha (TNF-alpha)) on whole blood samples. The results were compared with those of QFT. Regarding sensitivity in the TB group, patients in the QFT positive TB group (N = 22) were all ICCFC positive and 9 patients (64%) in the QFT negative TB group (N = 14) were ICCFC positive. In all test tubes (N = 72), sensitivity of "targeted" gating for TNF-alpha+ IFN-gamma+ CD4+ T cells was significantly higher than customary gating (72%, 54%, respectively, P = 0.001). The diagnostic sensitivity of ICCFC was further confirmed to be much higher than that of QFT. In the ICCFC analysis, TNF-alpha+ IFN-gamma+ CD4+ T cells should be sequentially gated through appropriately defined regions, minimizing interferents and reflecting characteristics of light scatter and marker expressions of CD4+ T cells activated by TB antigens.

In search of differences between the two types of sensory cells innervating spider slit sensilla (Cupiennius salei Keys.)

The metatarsal lyriform organ of the spider Cupiennius salei is a vibration detector consisting of 21 cuticular slits supplied by two sensory cells each, one ending in the outer and the other at the inner slit membrane. In search of functional differences between the two cell types due to differences in stimulus transmission, we analyzed (1) the adaptation of responses to electrical stimulation, (2) the thresholds for mechanical stimulation and (3) the representation of male courtship vibrations using intracellular recording and staining techniques. Single- and multi-spiking receptor neurons were found among both cell types, which showed high-pass filter characteristics. Below 100-Hz threshold, tarsal deflections were between 1 degrees and 10 degrees. At higher frequencies, they decreased down to values as small as 0.05 degrees, corresponding to 4.5-nm tarsal deflection in the most sensitive cases. Different slits in the organ and receptor cells with slow or fast adaptation did not differ in this regard. When stimulated with male courtship vibrations, both types of receptor cells again did not differ significantly regarding number of action potentials, latency and synchronization coefficients. Surprisingly, the differences in dendrite coupling were not reflected by the physiological responses of the two cell types innervating the slits.

Anatomy and physiology of a set of low‐frequency vibratory interneurons in a nonhearing ensiferan (Troglophilus neglectus, Rhaphidophoridae)

Vibratory interneurons were investigated in a primitive nonhearing ensiferan (orthopteran) species (Troglophilus neglectus, Rhaphidophoridae), using intracellular recording and staining technique. The study included 26 morphologically and/or physiologically distinct types of neurons from the prothoracic ganglion responding to vibration of the front legs. Most of these neurons are tuned to frequencies below 400 Hz. The morphology, anatomical position in the ganglion, and physiological responses are described in particular for a set of these low-frequency-tuned elements, including one local neuron, two T-shaped fibers, and five descending neurons, for which no putative homologues are known from the hearing Orthoptera. Their lowest thresholds are between about 0.01 and 0.4 m/second(2) at frequencies of 50-400 Hz, and the shortest latencies between 10 and 16 msec, suggesting that they are first- or second-order interneurons. Six interneurons have dendritic arborizations in the neuropile region that contains projections of tibial organ vibratory receptors, but their sensitivity suggests predominating inputs from vibrational sensilla of another origin. Responses of most neurons are composed of frequency-specific excitatory and inhibitory synaptic potentials, most of the latter being received in the high-frequency range. The function of these neurons in predator detection and intraspecific communication is discussed.

Harmonization of light scatter and fluorescence flow cytometry profiles obtained after staining peripheral blood leucocytes for cell surface‐only versus intracellular antigens with the Fix &amp; Perm™ reagent

Staining for intracellular markers with the Fix & Perm reagent is associated with variations in the scatter properties of leucocytes, limiting automated analysis of flow cytometry (FCM) data. Here, we investigated those variables significantly contributing to changes in the light scatter, autofluorescence, and bcl2 staining characteristics of peripheral blood (PB) leucocytes, after fixation with Fix & Perm. Our major aim was to evaluate a new mathematical approach for automated harmonization of FCM data from datafiles corresponding to aliquots of a sample treated with cell-surface-only versus Fix & Perm intracellular staining techniques. Overall, neither the anticoagulant used nor sample storage for <24 h showed significant impact on the light scatter and fluorescence properties of PB leucocytes; similarly, the duration of the fixation period (once >15 min were used) had a minimum impact on the FCM properties of PB leucocytes. Conversely, changes in cell/protein concentrations and the fixative/sample (vol/vol) ratio had a clear impact on the light scatter features of some populations of leucocytes. Accordingly, lower cell/protein concentrations were associated with lower scatter values, particularly for the neutrophils. Such changes could be partially corrected through the use of higher fixative to sample volume ratios. Despite the variable changes detected between aliquots of the same sample treated with cell surface-only versus intracellular staining procedures, the new mathematical approach here proposed and evaluated for automated harmonization of common parameters in both datafiles, could correct the FCM profiles of leucocytes derived from cells undergoing conventional fixation/permeabilization procedures, and made them indistinguishable from those corresponding to aliquots of the same sample treated with cell-surface-only staining techniques.

Neural pathways for the processing of alarm pheromone in the ant brain

Social insects like ants exhibit sophisticated communication by means of pheromones, one example of which is the use of alarm pheromones to alert nestmates for colony defense. In the ant Camponotus obscuripes, we have reported that information about formic acid and n-undecane, alarm pheromone components, is processed in a set of specific glomeruli in the antennal lobe (primary olfactory center). Alarm pheromone signals are then transmitted, mainly via uniglomerular projection neurons (uni-PNs), to the protocerebrum (PR), where sensory signals are integrated to form motor commands for behavioral responses. In this study, we physiologically and morphologically characterized 63 alarm pheromone-sensitive PR neurons in ants by using intracellular recording and staining techniques. Most of the pheromone-sensitive PR neurons had dendrites in the mushroom body (MB), the lateral horn, or the medial PR. Some neurons with dendrites in these areas responded specifically to formic acid or n-undecane and may participate in the control of specific behavioral responses to each pheromone component. Other neurons responded also to non-pheromonal odors, in contrast to uni-PNs, most of which responded specifically to alarm pheromones. Responses to non-pheromonal odors were most prominent in efferent neurons of the MB lobe, suggesting that they may participate in integration of pheromonal and non-pheromonal information. We found a class of PR neurons that receives input in all of these pheromone-processing areas and terminates in a variety of premotor areas. These neurons may participate in the control of pheromone-sensitized aggressive behavior, which is triggered by non-pheromonal sensory stimuli associated with a potential enemy.

Detection by Flow Cytometry of ESAT-6- and PPD-Specific Circulating CD4+ T Lymphocytes as a Diagnostic Tool for Tuberculosis

Background: The identification of mycobacteria represents the gold standard in the diagnosis of tuberculosis (TB), but it is not applicable in all patients, and immunological tests, such as the tuberculin skin test (TST), are not specific and sensitive enough. Methods: By flow cytometry, we measured the CD4 T-cell response to purified protein derivative (PPD) and early secretory antigenic target-6 (ESAT-6) protein using the intracellular cytokine staining technique on whole blood samples obtained from active TB (n = 16), latent TB (n = 17), Bacille Calmette-Guérin (BCG)-vaccinated (n = 11) and healthy (n = 10) donors. All the patients were also tested with conventional TST. Results: The identification by flow cytometry of PPD-specific T lymphocytes upon antigen stimulation of whole blood enables the discrimination of active TB, latent TB and BCG-vaccinated subjects from healthy individuals, whereas the ESAT-6 response discriminated active TB from healthy and BCG-vaccinated individuals. Moreover, this test enables identification of active TB patients who were negative on TST and to distinguish between TB and non-typical mycobacteria TB infections. Conclusions: The identification by flow cytometry of antigen-specific T lymphocytes upon antigen stimulation of whole blood has a better positive predictive value than TST, and could represent a further tool in the diagnosis of TB infection.

Pheromone-sensitive glomeruli in the primary olfactory centre of ants

Tremendous evolutional success and the ecological dominance of social insects, including ants, termites and social bees, are due to their efficient social organizations and their underlying communication systems. Functional division into reproductive and sterile castes, cooperation in defending the nest, rearing the young and gathering food are all regulated by communication by means of various kinds of pheromones. No brain structures specifically involved in the processing of non-sexual pheromone have been physiologically identified in any social insects. By use of intracellular recording and staining techniques, we studied responses of projection neurons of the antennal lobe (primary olfactory centre) of ants to alarm pheromone, which plays predominant roles in colony defence. Among 23 alarm pheromone-sensitive projection neurons recorded and stained in this study, eight were uniglomerular projection neurons with dendrites in one glomerulus, a structural unit of the antennal lobe, and the remaining 15 were multiglomerular projection neurons with dendrites in multiple glomeruli. Notably, all alarm pheromone-sensitive uniglomerular projection neurons had dendrites in one of five 'alarm pheromone-sensitive (AS)' glomeruli that form a cluster in the dorsalmost part of the antennal lobe. All alarm pheromone-sensitive multiglomerular projection neurons had dendrites in some of the AS glomeruli as well as in glomeruli in the anterodorsal area of the antennal lobe. The results suggest that components of alarm pheromone are processed in a specific cluster of glomeruli in the antennal lobe of ants.