John Dempster

In situ characterization of CD4 T cell behavior in mucosal and systemic lymphoid tissues during the induction of oral priming and tolerance

The behavior of antigen-specific CD4+ T lymphocytes during initial exposure to antigen probably influences their decision to become primed or tolerized, but this has not been examined directly in vivo. We have therefore tracked such cells in real time, in situ during the induction of oral priming versus oral tolerance. There were marked contrasts with respect to rate and type of movement and clustering between naive T cells and those exposed to antigen in immunogenic or tolerogenic forms. However, the major difference when comparing tolerized and primed T cells was that the latter formed larger and longer-lived clusters within mucosal and peripheral lymph nodes. This is the first comparison of the behavior of antigen-specific CD4+ T cells in situ in mucosal and systemic lymphoid tissues during the induction of priming versus tolerance in a physiologically relevant model in vivo.

The properties of 5-HT3 receptors in clonal cell lines studied by patch-clamp techniques

1 The characteristics of transmembrane currents evoked by 5‐hydroxytryptamine (5‐HT) in the neuroblastoma x Chinese hamster brain cell line NCB‐20 and neuroblastoma clonal cell line N1E‐115 have been studied under voltage‐clamp conditions by the whole‐cell recording and outside‐out membrane patch modes of the patch‐clamp technique. 2 In 73% of NCB‐20 cells examined (n = 221), and all N1E‐115 cells studied (n = 80), 5‐HT (10 μm) elicited a transient inward current at negative holding potentials, this being associated with an increase in membrane conductance. In both cell lines responses to 5‐HT reversed in sign at a potential of approximately −2mV and demonstrated inward rectification. 3 The reversal potential of 5‐HT‐induced currents (E5‐HT) recorded from either NCB‐20 or N1E‐115 cells was unaffected by total replacement of internal K+ by Cs+. In N1E‐115 cells, reducing internal K+ concentration from 140 to 20 mm produced a positive shift in E5‐HT of approximately 28 mV, whereas reducing external Na+ from 143 to 20 mm was associated with a negative shift in E5‐HT of about 37 mV. A large reduction in internal Cl− concentration (from 144 to 6 mm) had little effect on E5‐HT. 4 5‐HT‐induced currents of NCB‐20 cells were unaffected by methysergide (1 μm) or ketanserin (1 μm), but were reversibly antagonized by GR38032F (0.1–1.0 nm) with an IC50 of 0.25 nm. GR 38032F (0.3 nm) reduced 5‐HT‐induced currents in N1E‐115 cells to approximately 26% of their control value. 5 On outside‐out membrane patches excised from both NCB‐20 and N1E‐115 cells, 5‐HT induced small inward currents which could not be clearly resolved into discrete single channel events. Such responses were: (i) reversibly antagonized by GR 38032F (1 nm) (ii) reversed in sign at 0 mV, and (iii) subject to desensitization. 6 Fluctuation analysis of inward currents evoked by 5‐HT (1 μm) in N1E‐115 cells suggests that 5‐HT gates a channel with a conductance of approximately 310fS. Such a relatively small conductance could readily explain why the response of outside‐out membrane patches to 5‐HT cannot at present be resolved into clear single channel events.

Cutting edge: acute lung allograft rejection is independent of secondary lymphoid organs

It is the prevailing view that adaptive immune responses are initiated in secondary lymphoid organs. Studies using alymphoplastic mice have shown that secondary lymphoid organs are essential to initiate allograft rejection of skin, heart, and small bowel. The high immunogenicity of lungs is well recognized and allograft rejection remains a major contributing factor to poor outcomes after lung transplantation. We show in this study that alloreactive T cells are initially primed within lung allografts and not in secondary lymphoid organs following transplantation. In contrast to other organs, lungs are acutely rejected in the absence of secondary lymphoid organs. Two-photon microscopy revealed that recipient T cells cluster predominantly around lung-resident, donor-derived CD11c+ cells early after engraftment. These findings demonstrate for the first time that alloimmune responses following lung transplantation are initiated in the graft itself and therefore identify a novel, potentially clinically relevant mechanism of lung allograft rejection.

GABAA and glycine receptor mediated transmission in rat lamina II neurones: relevance to the analgesic actions of neuroactive steroids

Analgesic neurosteroids such as 5α‐pregnan‐3α‐ol‐20‐one (5α3α) are potent selective endogenous modulators of the GABAA receptor (GABAAR) while certain synthetic derivatives (i.e. minaxolone) additionally enhance the function of recombinant glycine receptors (GlyR). Inhibitory transmission within the superficial dorsal horn has been implicated in mediating the analgesic actions of neurosteroids. However, the relative contribution played by synaptic and extrasynaptic receptors is unknown. In this study, we have compared the actions of 5α3α and minaxolone upon inhibitory transmission mediated by both GABAA and strychnine‐sensitive GlyRs in lamina II neurones of juvenile (P15–21) rats. At the near physiological temperature of 35°C and at a holding potential of −60 mV we recorded three kinetically distinct populations of miniature IPSCs (mIPSCs): GlyR‐mediated, GABAAR‐mediated and mixed GABAAR‐GlyR mIPSCs, arising from the corelease of both inhibitory neurotransmitters. In addition, sequential application of strychnine and bicuculline revealed a small (5.2 ± 1.0 pA) GlyR‐ but not a GABAAR‐mediated tonic conductance. 5α3α (1–10 μm) prolonged GABAAR and mixed mIPSCs in a concentration‐dependent manner but was without effect upon GlyR mIPSCs. In contrast, minaxolone (1–10 μm) prolonged the decay of GlyR mIPSCs and, additionally, was ∼10‐fold more potent than 5α3α upon GABAAR mIPSCs. However, 5α3α and minaxolone (1 μm) evoked a similar bicuculline‐sensitive inhibitory conductance, indicating that the extrasynaptic GABAARs do not discriminate between these two steroids. Furthermore, ∼92% of the effect of 1 μm 5α3α upon GABAergic inhibition could be accounted for by its action upon the extrasynaptic conductance. These findings are relevant to modulation of inhibitory circuits within spinally mediated pain pathways and suggest that extrasynaptic GABAARs may represent a relevant molecular target for the analgesic actions of neurosteroids.

Selective release of molecules from weibel-palade bodies during a lingering kiss

Exocytosis of specialized endothelial cell secretory organelles, Weibel-Palade bodies (WPBs), is thought to play an important role in regulating hemostasis and intravascular inflammation. The major WPB core proteins are Von Willebrand factor (VWF) and its propolypeptide (Proregion), constituting more than 95% of the content. Although the composition of the WPBs can be fine-tuned to include cytokines and chemokines (eg, interleukin-8 [IL-8] and eotaxin-3), it is generally assumed that WPB exocytosis is inextricably associated with secretion of VWF. Here we show that WPBs can undergo a form of exocytosis during which VWF and Proregion are retained while smaller molecules, such as IL-8, are released. Imaging individual WPBs containing fluorescent cargo molecules revealed that during weak stimulation approximately 25% of fusion events result in a failure to release VWF or Proregion. The WPB membrane protein P-selectin was also retained; however, the membrane tetraspannin CD63 was released. Accumulation or exclusion of extracellular fluorescent dextran molecules ranging from 3 kDa to 2 mDa show that these events arise due to the formation of a fusion pore approximately 12 nm in diameter. The pore behaves as a molecular filter, allowing selective release of WPB core and membrane proteins. WPB exocytosis is not inextricably associated with secretion of VWF.

Nicotinic antagonist-produced frequency-dependent changes in acetylcholine release from rat motor nerve terminals

1. The frequency (0.5‐150 Hz) and calcium dependence (0.5‐2.0 mM) of the effects of the nicotinic antagonist tubocurarine (0.2 microM) on acetylcholine (ACh) liberation from motor nerve terminals has been examined using binomial analysis of quantal transmitter release. 2. At an extracellular calcium ion concentration ([Ca2+]o) of 2.0 mM, tubocurarine produced a decrease in the endplate current (EPC) quantal content of approximately 30% at high frequencies of motor nerve stimulation (50‐150 Hz). In contrast, at low frequencies of stimulation (0.5‐1.0 Hz), tubocurarine enhanced the EPC quantal content by approximately 20%. 3. The enhancement of EPC quantal content produced by tubocurarine at low frequencies of motor nerve stimulation was [Ca2+]o dependent, being abolished when [Ca2+]o was lowered from 2.0 to 0.5 mM. In contrast, the decrease in quantal content produced by tubocurarine at high frequencies of motor nerve stimulation was independent of [Ca2+]o, being approximately 30% at all calcium ion concentrations studied. 4. In direct contrast to tubocurarine, the nicotinic antagonist vecuronium (1.0 microM) produced no increase in EPC quantal content at low frequencies of nerve stimulation. However, at high frequencies of nerve stimulation it decreased EPC quantal content to a similar extent to 0.2 microM tubocurarine. The frequency‐dependent decrease in EPC quantal content produced by 1.0 microM vecuronium in 2.0 mM [Ca2+]o was very similar to that seen with 0.2 microM tubocurarine in 0.5 mM [Ca2+]o. 5. Binomial analysis revealed that all the changes in EPC quantal content associated with both nicotinic antagonists were due to changes in the size of the pool of quanta in the nerve terminal available for immediate release with no effect on the probability of release of an individual quantum. 6. The results are interpreted in terms of two separately identifiable prejunctional actions of the nicotinic antagonists, both involving an action at nicotinic ACh receptors situated on the motor nerve terminal. Thus, at high frequencies of motor nerve stimulation tubocurarine and vecuronium produce a [Ca2+]o‐independent decrease in ACh release, probably through an inhibitory action on a positive‐feedback prejunctional nicotinic autoreceptor closely related to the muscle‐type nicotinic ACh autoreceptor. However, at low frequencies of motor nerve stimulation we suggest that tubocurarine, but not vecuronium, produces a [Ca2+]o‐dependent increase in ACh release through an action at a negative‐feedback prejunctional neuronal‐type nicotinic ACh autoreceptor.

Chapter 16. Two-photon microscopy and multidimensional analysis of cell dynamics.

Two-photon (2P) microscopy is a high-resolution imaging technique that was initially applied by neurobiologists and developmental cell biologists but has subsequently been broadly adapted by immunologists. The value of 2P microscopy is that it affords an unparalleled view of single-cell spatiotemporal dynamics deep within intact tissues and organs. As the technology develops and new transgenic mice and fluorescent probes become available, 2P microscopy will serve as an increasingly valuable tool for assessing cell function and probing molecular mechanisms. Here we discuss the technical aspects related to 2P microscope design, explain in detail various tissue imaging preparations, and walk the reader through the often daunting process of analyzing multidimensional data sets and presenting the experimental results.

Importance of vagally mediated bradycardia for the induction of torsade de pointes in an in vivo model.

Bradycardia is a risk factor for the development of torsade de pointes (TdP). The aim of this work was to compare the importance of changes in heart rate and arterial blood pressure in the development of drug‐induced TdP and to investigate the role of vagal influences.

Pharmacological characterization of a rat 5‐hydroxytryptamine type3 receptor subunit (r5‐HT3A(b)) expressed in Xenopus laevis oocytes

The present study has utilized the two electrode voltage‐clamp technique to examine the pharmacological profile of a splice variant of the rat orthologue of the 5‐hydroxytryptamine type 3A subunit (5‐HT3A(b)) heterologously expressed in Xenopus laevis oocytes. At negative holding potentials, bath applied 5‐HT (300 nM–10 μM) evoked a transient, concentration‐dependent (EC50=1.1±0.1 μM), inward current. The response reversed in sign at a holding potential of −2.1±1.6 mV. The response to 5‐HT was mimicked by the 5‐HT3 receptor selective agonists 2‐methyl‐5‐HT (EC50=4.1±0.2 μM), 1‐phenylbiguanide (EC50=3.0±0.1 μM), 3‐chlorophenylbiguanide (EC50=140± 10 nM), 3,5‐dichlorophenylbiguanide (EC50=14.5±0.4 nM) and 2,5‐dichlorophenylbiguanide (EC50= 10.2±0.6 nM). With the exception of 2‐methyl‐5‐HT, all of the agonists tested elicited maximal current responses comparable to those produced by a saturating concentration (10 μM) of 5‐HT. Responses evoked by 5‐HT at EC50 were blocked by the 5‐HT3 receptor selective antagonist ondansetron (IC50=231±22 pM) and by the less selective agents (+)‐tubocurarine (IC50=31.9± 0.01 nM) and cocaine (IC50=2.1±0.2 μM). The data are discussed in the context of results previously obtained with the human and mouse orthologues of the 5‐HT3A subunit. Overall, the study reinforces the conclusion that species differences detected for native 5‐HT3 receptors extend to, and appear largely explained by, differences in the properties of homo‐oligomeric receptors formed from 5‐HT3A subunit orthologues.

Potentiation of E-4031-induced torsade de pointes by HMR1556 or ATX-II is not predicted by action potential short-term variability or triangulation

Torsade de pointes (TdP) can be induced by a reduction in cardiac repolarizing capacity. The aim of this study was to assess whether I Ks blockade or enhancement of I Na could potentiate TdP induced by I Kr blockade and to investigate whether short‐term variability (STV) or triangulation of action potentials preceded TdP.