Alpha Beta Gamma Delta Research Articles

Transcription Factor Gene Fluorescent Reporters Track Lineage Fate in Lymphoid Commitment

The transition from multipotency to lineage commitment can be followed with particular clarity for T cell precursors. In this lineage, the role of environmental signals can be clearly separated from the role of intrinsic fate programming in individual cells and the cells' developmental responses to changing conditions and can be tracked in real time. T cell precursors are still multipotent when they first enter the thymus, and if they are removed from the thymic microenvironment at this stage they can give rise to non-T cells including dendritic cells and myeloid cells. For multiple cell divisions, they preserve this multipotency and are only kept in line to become T cells conditionally, by Notch signaling from the thymic stroma. Then at a specific point of no return, the cells become unable to give rise to anything except T cells regardless of environment, and this is the point of commitment. Commitment is clearly the readout of a change in internal transcriptional regulatory state. To determine how this is controlled, we and others have charted transcription factor expression changes across this interval and changes in chromatin modification and DNA accessibility that accompany the transition, and we have been able to use functional perturbation tests to narrow down the key regulators that catalyze and enforce this transition. A particularly important commitment factor is encoded by the Bcl11b gene, which is released from previously repressed chromatin and sharply activated at the transcriptional level just as the cells become committed. The Bcl11b gene product is required in all alpha beta and most gamma delta T cells to enable the commitment process to occur. These properties make it highly illuminating as an indicator of the regulatory state in individual differentiating T-cell precursors. We have generated a series of knock-in Bcl11b fluorescent reporter alleles to probe the correlation of Bcl11b expression with changes in specific target genes, to determine the transcription factor requirements for Bcl11b gene activation in the gene regulatory network controlling commitment in single cells, and to measure the role of epigenetic modification of the Bcl11b locus on the kinetics of transition from uncommitted to committed states. These results and their implications will be presented. Importantly, the use of live-cell reporters reveals a level of all-or-none, stochastic regulation in the responses of individual cells to combinatorial transcription factor action at this developmental watershed1.Reference:1. Kueh HY, Yui MA, Ng KKH, et al. Asynchronous combinatorial action of four regulatory factors activates Bcl11b for T cell commitment. Nature Immunology. 2016.17, 956-965. DisclosuresNo relevant conflicts of interest to declare.

Pharmacological characterization of a novel positive modulator at α4β3δ-containing extrasynaptic GABAA receptors

The in vitro and in vivo pharmacological effects of [2-amino-4-(2,4,6-trimethylbenzylamino)-phenyl]-carbamic acid ethyl ester (AA29504), which is a close analogue of retigabine, have been investigated. AA29504 induced a rightward shift of the activation threshold at cloned KCNQ2, 2/3 and 4 channels expressed in Xenopus oocytes, with a potency 3-4fold lower than retigabine. AA29504 (1 muM) had no agonist activity when tested at alpha(1)beta(3)gamma(2s) or alpha(4)beta(3)delta GABA(A) receptors expressed in Xenopus oocytes, but left-shifted the EC(50) for GABA and gaboxadol (THIP) at both receptors. The maximum GABA response at alpha(1)beta(3)gamma(2s) receptors was unchanged by AA29504 (1 muM), but increased 3-fold at alpha(4)beta(3)delta receptors. In slices prepared from the prefrontal cortex of adult rats AA29504 had no effect alone on the average IPSC or the tonic current in layer II/III pyramidal neurons, but potentiated the effect of gaboxadol on both phasic and tonic currents. Thus, the effects of gaboxadol could be positively modulated by AA29504. Systemic administration of AA29504 at doses relevant for modulating GABA transmission produced anxiolytic effects and reduced motor coordination consistent with activity at GABA(A) receptors. We conclude that AA29504 exerts a major action via alpha(4)beta(3)delta-containing GABA(A) receptors, which will be important for interpreting its effect in vivo.

Killing of human leukaemia/lymphoma B cells by activated cytotoxic T lymphocytes in the presence of a bispecific monoclonal antibody (alpha CD3/alpha CD19).

Bispecific antibodies (BsAb) can be used to retarget T cells irrespective of their specificity to certain target cells inducing target cell lysis. We have tested the efficacy of the BsAb SHR-1, directed against the T cell antigen CD3 and the B cell antigen CD19 to induce (malignant) B cell kill by T cells as measured in a 51Cr-release assay. Two cytotoxic T cell clones (CTL), expressing TCR alpha beta or TCR gamma delta, were effective in killing CD19 expressing B cell lines at different stages of differentiation in the presence, but not in the absence, of the BsAb. CD19- target cells were not killed. Fresh CD19+ leukaemia/lymphoma cells were also efficiently killed by SHR-1 preincubated CTL clones. In addition, phytohaemagglutinin (PHA) or CD3-activated IL-2 expanded peripheral blood mononuclear cells (PBMC) of normal donors did so after 2 weeks of stimulation. A concentration of 100 ng/ml of the BsAb was sufficient to obtain optimal lysis of all target cells tested. These results show that fresh human leukaemia/lymphoma cells, freshly derived from active lymphoblastic leukaemia (ALL) as well as non-Hodgkin's lymphoma (NHL) patients, can be effectively killed in the presence of this BsAb by activated T cells.

Antigen‐specific Apheresis of Pathogenic Autoantibodies from Myasthenia Gravis Sera

Myasthenia gravis (MG) is usually caused by autoantibodies against muscle nicotinic acetylcholine receptor (AChR), which is composed of five subunits (alpha(2)betagammadelta or alpha(2)betaepsilondelta). Current treatments, including plasmapheresis, are nonspecific, causing several side effects. We aim to develop an antigen-specific alternative to plasmapheresis, since the latter removes indispensable plasma components in addition to anti-AChR antibodies. We are developing a method for the selective depletion of the anti-AChR autoantibodies from patients' plasma through the construction of "immunoadsorbent" columns carrying AChR domains. We have expressed the extracellular domains (ECDs, amino acids approximately 1-210/220) of all human muscle AChR subunits in Pichia pastoris and, in preliminary experiments, in E. coli. The ECDs were immobilized (individually or mixed) on Sepharose beads, producing Sepharose-ECD columns, which were tested for their immunoadsorbing capacity on MG sera and shown to specifically eliminate major autoantibody fractions from several MG sera. The immobilized ECDs remained stable and did not dissociate from their matrix after incubation with serum, whereas the procedure was neither toxic nor immunogenic in two experimental rabbits. Testing the intact or antibody-depleted MG sera and the affinity purified autoantibodies showed that both the intact sera and the purified autoantibodies, but not the antibody-depleted sera, could induce AChR loss in cell cultures and experimental MG in rats. This preliminary study suggests that the myasthenic potency of MG sera is entirely due to their anti-AChR antibodies and therefore their depletion should be of therapeutic value. We conclude that ECD-mediated immunoadsorption can be used as an efficient, antigen-specific therapy for MG.

RNA backbone: consensus all-angle conformers and modular string nomenclature (an RNA Ontology Consortium contribution).

A consensus classification and nomenclature are defined for RNA backbone structure using all of the backbone torsion angles. By a consensus of several independent analysis methods, 46 discrete conformers are identified as suitably clustered in a quality-filtered, multidimensional dihedral angle distribution. Most of these conformers represent identifiable features or roles within RNA structures. The conformers are given two-character names that reflect the seven-angle delta epsilon zeta alpha beta gamma delta combinations empirically found favorable for the sugar-to-sugar "suite" unit within which the angle correlations are strongest (e.g., 1a for A-form, 5z for the start of S-motifs). Since the half-nucleotides are specified by a number for delta epsilon zeta and a lowercase letter for alpha beta gamma delta, this modular system can also be parsed to describe traditional nucleotide units (e.g., a1) or the dinucleotides (e.g., a1a1) that are especially useful at the level of crystallographic map fitting. This nomenclature can also be written as a string with two-character suite names between the uppercase letters of the base sequence (N1aG1gN1aR1aA1cN1a for a GNRA tetraloop), facilitating bioinformatic comparisons. Cluster means, standard deviations, coordinates, and examples are made available, as well as the Suitename software that assigns suite conformer names and conformer match quality (suiteness) from atomic coordinates. The RNA Ontology Consortium will combine this new backbone system with others that define base pairs, base-stacking, and hydrogen-bond relationships to provide a full description of RNA structural motifs.

Novel α‐conotoxins from Conus spurius and the α‐conotoxin EI share high‐affinity potentiation and low‐affinity inhibition of nicotinic acetylcholine receptors

alpha-Conotoxins from marine snails are known to be selective and potent competitive antagonists of nicotinic acetylcholine receptors. Here we describe the purification, structural features and activity of two novel toxins, SrIA and SrIB, isolated from Conus spurius collected in the Yucatan Channel, Mexico. As determined by direct amino acid and cDNA nucleotide sequencing, the toxins are peptides containing 18 amino acid residues with the typical 4/7-type framework but with completely novel sequences. Therefore, their actions (and that of a synthetic analog, [gamma15E]SrIB) were compared to those exerted by the alpha4/7-conotoxin EI from Conus ermineus, used as a control. Their target specificity was evaluated by the patch-clamp technique in mammalian cells expressing alpha(1)beta(1)gammadelta, alpha(4)beta(2) and alpha(3)beta(4) nicotinic acetylcholine receptors. At high concentrations (10 microm), the peptides SrIA, SrIB and [gamma15E]SrIB showed weak blocking effects only on alpha(4)beta(2) and alpha(1)beta(1)gammadelta subtypes, but EI also strongly blocked alpha(3)beta(4) receptors. In contrast to this blocking effect, the new peptides and EI showed a remarkable potentiation of alpha(1)beta(1)gammadelta and alpha(4)beta(2) nicotinic acetylcholine receptors if briefly (2-15 s) applied at concentrations several orders of magnitude lower (EC(50), 1.78 and 0.37 nm, respectively). These results suggest not only that the novel alpha-conotoxins and EI can operate as nicotinic acetylcholine receptor inhibitors, but also that they bind both alpha(1)beta(1)gammadelta and alpha(4)beta(2) nicotinic acetylcholine receptors with very high affinity and increase their intrinsic cholinergic response. Their unique properties make them excellent tools for studying the toxin-receptor interaction, as well as models with which to design highly specific therapeutic drugs.

DYSREGULATON OF CYTOKINES BY GAMMA DELTA T CELLS IN CHILDREN WITH CYSTIC FIBROSIS AND ITS CORRELATON WITH DISEASE STATE

PURPOSE: Role of gamma delta T cells at the pulmonary level in children with CF remains unexplored. We studied the invitro intracellular cytokine expression in children with CF and normal controls to correlate the cytokine expression by alpha beta gamma delta T-cells with the clinical status.

The Tmc Complex fromDesulfovibrio vulgarisHildenborough Is Involved in Transmembrane Electron Transfer from Periplasmic Hydrogen Oxidation

Three membrane-bound redox complexes have been reported in Desulfovibrio spp., whose genes are not found in the genomes of other sulfate reducers such as Desulfotalea psycrophila and Archaeoglobus fulgidus. These complexes contain a periplasmic cytochrome c subunit of the cytochrome c(3) family, and their presence in these organisms probably correlates with the presence of a pool of periplasmic cytochromes c(3), also absent in the two other sulfate reducers. In this work we report the isolation and characterization of the first of such complexes, Tmc from D. vulgaris Hildenborough, which is associated with the tetraheme type II cytochrome c(3). The isolated Tmc complex contains four subunits, including the TpIIc(3) (TmcA), an integral membrane cytochrome b (TmcC), and two cytoplasmically predicted proteins, an iron-sulfur protein (TmcB) and a tryptophan-rich protein (TmcD). Spectroscopic studies indicate the presence of eight hemes c and two hemes b in the complex pointing to an alpha(2)betagammadelta composition (TmcA(2)BCD). EPR analysis reveals the presence of a [4Fe4S](3+) center and up to three other iron-sulfur centers in the cytoplasmic subunit. Nearly full reduction of the redox centers in the Tmc complex could be obtained upon incubation with hydrogenase/TpIc(3), supporting the role of this complex in transmembrane transfer of electrons resulting from periplasmic oxidation of hydrogen.

The Human Leukocyte Antigen (HLA) System

The discovery of Major Histocompatibility Complex (MHC) and its involvement in graft rejection, immune response and the genetic basis of disease associations lead to the birth of this new field of science called Immunogenetics. This field is important not only in basic biomedical research but also in clinical medicine. The growth of this field was further substantiated by the various International Histocompatibility Workshops. Solutions to idiopathic syndromes and autoimmune diseases come from the field of Immunology, Immunogenetics and Molecular biology. In studying the genetic basis of disease susceptibility in human beings, one has to have a different approach because predictive experimental mating cannot be achieved in humans as in animal models; moreover the generation time is longer. One approach is through a study of randomly selected patients and comparing their results with those of controls and the other is through a study of affected families (nuclear or extended) and looking for the mode of inheritance of the disease with relation to genetic markers. Nonetheless the sampling stratification, sample heterogeneity, disease heterogeneity, and age at onset, epidemiological conditions and other socio-biological factors limit these studies. Essentially attempts are to be made to control these parameters and achieve a meaningful conclusion. The current concepts of antigen presentation to immunocompetent cells indicate that antigen processing takes place in the acid environment of the endosomes of antigen presenting cells. Proteolytic degradation of the antigenic proteins results in peptides of different fragments, which are subsequently presented by MHC molecules after they are bound to peptide binding site of the MHC molecules which can bind a variety of peptides having in common, a peptide backbone. These immunogenic peptides (held snugly in the groove by hydrogen bonds between the MHC protein and the peptide backbone) on antigen presenting cells, with the MHC molecule and T-cell receptor on T-cells form a stable trimolecular complex. Thus MHC molecules and alpha beta T-cell receptors or gamma delta T-cell receptor play a major role in the subsequent immune response. The polymorphism of the MHC molecules and T-cell receptor may play an important role in the antigen recognition process. It is possible that a few of the many alleles of a given HLA locus may provide a more stable trimolecular complex than others, thus resulting in a ‘high responder status’ of an individual than the rest of the alleles. This field of HLA system has been evolving very fast and therefore an updated HLA system review has been provided.

Immunogenicity of Torpedo acetylcholine receptor in the context of different rat MHC class II haplotypes and non-MHC genomes.

The nicotinic acetylcholine receptor (nAChR) is the autoantigen in seropositive myasthenia gravis (MG), a T-cell-dependent B-cell-mediated autoimmune disease. The nAChR is a pentameric transmembrane receptor comprising alpha alpha beta gamma delta chains. During early postnatal development the nAChR gamma chain is replaced by the nAChR epsilon chain. We tested the myasthenogenicity in experimental autoimmune myasthenia gravis (EAMG) of the native nAChR derived from the electric ray Torpedo californica (T-nAChR) in various inbred and MHC -congenic rat strains. Differences in the disease course emerged dependent on the MHC haplotype and non-MHC genes. Interestingly, no tested rat strain was completely resistant to EAMG, but there were strong differences in disease severity mainly depending on the MHC haplotype. In the LEW non-MHC genome, the B-cell response and the severity of EAMG were dependent on the expressed MHC haplotype. This study underscores the influence of genetic factors on disease severity, disease course and on the degree of the emerging antibody responses in EAMG.

Regulation of nicotinic acetylcholine receptor assembly.

The four muscle-type nicotinic acetylcholine receptor (AChR) subunits, alpha, beta, gamma, and delta, assemble into functional alpha(2)betagammadelta pentamers in the endoplasmic reticulum (ER) through a series of interdependent folding and oligomerization events. The first stable assembly intermediate is a trimer composed of alpha, beta, and gamma subunits. The formation of alphabetagamma trimers initiates a series of subunit folding and processing events that allow addition of delta subunits to form alphabetagammadelta tetramers. Subunit folding and processing continue with formation of the ligand-binding sites on the alpha subunit of alphabetagammadelta tetramers and the second alpha subunit added to assemble alpha(2)betagammadelta pentamers. AChR assembly is inefficient. Only 20-30% of synthesized subunits assemble into mature receptors in the ER, while the remaining unassembled subunits are degraded. However, the efficiency of subunit assembly can be regulated under certain conditions leading to higher AChR expression. Increased intracellular cAMP levels cause a 2- to 3-fold increase in AChR assembly efficiency and a comparable increase in surface expression. Additionally, block of ubiquitin-proteasome degradation appears to enhance AChR assembly and expression. Thus, the regulation of AChR assembly through posttranslational mechanisms is a potential therapeutic target for increasing AChR expression in diseases in which expression is compromised.

A new iron oxidase from a moderately thermophilic iron oxidizing bacterium strain TI‐1

Iron oxidase was purified from plasma membranes of a moderately thermophilic iron oxidizing bacterium strain TI-1 in an electrophoretically homogeneous state. Spectrum analyses of purified enzyme showed the existence of cytochrome a, but not cytochrome b and c types. Iron oxidase was composed of five subunits with apparent molecular masses of 46 kDa (alpha), 28 kDa (beta), 24 kDa (gamma), 20 kDa (delta), and 17 kDa (epsilon). As the molecular mass of a native enzyme was estimated to be 263 kDa in the presence of 0.1% n-dodecyl-beta-D-maltopyranoside (DM), a native iron oxidase purified from strain TI-1 seems to be a homodimeric enzyme (alpha beta gamma delta epsilon)(2). Optimum pH and temperature for iron oxidation were pH 3.0 and 45 degrees C, respectively. The K(m) of iron oxidase for Fe(2+) was 1.06 mM and V(max) for O(2) uptake was 13.8 micromol x mg(-1) x min(-1). The activity was strongly inhibited by cyanide and azide. Purified enzyme from strain TI-1 is a new iron oxidase in which electrons of Fe(2+) were transferred to haem a and then to the molecular oxygen.

Inaugural Article: Acetylcholine receptor channel structure in the resting, open, and desensitized states probed with the substituted-cysteine-accessibility method

The nicotinic acetylcholine (ACh) receptors cycle among classes of nonconducting resting states, conducting open states, and nonconducting desensitized states. We previously probed the structure of the mouse-muscle ACh receptor channel in the resting state obtained in the absence of agonist and in the open states obtained after brief exposure to ACh. We now have probed the structure in the stable desensitized state obtained after many minutes of exposure to ACh. Muscle-type receptor has the subunit composition alpha(2)betagammadelta. Each subunit has four membrane-spanning segments, M1-M4. The channel lumen in the membrane domain is lined largely by M2 and to a lesser extent by M1 from each of the subunits. We determined the rates of reaction of a small, sulfhydryl-specific, charged reagent, 2-aminoethyl methanethiosulfonate with cysteines substituted for residues in alphaM2 and the alphaM1-M2 loop in the desensitized state and compared these rates to rates previously obtained in the resting and open states. The reaction rates of the substituted cysteines are different in the three functional states of the receptor, indicating significant structural differences. By comparing the rates of reaction of extracellularly and intracellularly added 2-aminoethyl methanethiosulfonate, we previously located the closed gate in the resting state between alphaG240 and alphaT244, in the predicted M1-M2 loop at the intracellular end of M2. Now, we have located the closed gate in the stable desensitized state between alphaG240 and alphaL251. The gate in the desensitized state includes the resting state gate and an extension further into M2.

The epitope detected by cytotoxic T lymphocytes against thymus leukemia (TL) antigen is TAP independent.

Thymus leukemia (TL) antigens belong to the family of MHC class Ib antigens. We have shown in our previous studies that they serve as transplantation antigens, and can be recognized by both TCR alpha beta and TCR gamma delta cytotoxic T lymphocytes (CTL) with TL but not H-2 restriction. Although TL are known to be expressed TAP independently, it is unclear whether peptide loading on TL molecules is necessary for the formation of CTL epitopes. In the present study, we first showed that TL expression is beta(2)-microglobulin (beta(2)m)-dependent but TAP1 independent by flow cytometric analysis of thymocytes from beta(2)m- or TAP1-deficient mice crossed with TL transgenic mice expressing Tla(a)-3-TL on their thymocytes. Subsequently, we investigated the epitope recognized by CTL derived from C3H mice immunized with skin from a transgenic mouse expressing T3(b)-TL ubiquitously. Bulk CTL lines against TL from primary mixed lymphocyte cultures showed comparable cytotoxicity against T3(b)-TL transfectants of TAP2-deficient murine RMA-S grown at 37 degrees C to that against those grown at 25 degrees C. Furthermore, TCR alpha beta and TCR gamma delta CTL clones against TL recognized TL expressed on T3(b)-TL transfectants of RMA-S and Drosophila melanogaster cells having broad defects in peptide loading of MHC, and lysed these target cells. These results together indicate that TL-specific CTL populations primarily recognize epitopes expressed TAP independently.

On the prosthetic groups of the NiFe sulfhydrogenase from Pyrococcus furiosus: topology, structure, and temperature-dependent redox chemistry.

The sulfhydrogenase complex of Pyrococcus furiosus is an alpha beta gamma delta heterotetramer with both hydrogenase activity (borne by the alpha delta subunits) and sulfur reductase activity (carried by the beta gamma subunits). The beta-subunit contains at least two [4Fe-4S] cubanes and the gamma-subunit contains one [2Fe-2S] cluster and one FAD molecule. The delta-subunit contains three [4Fe-4S] cubanes and the alpha-subunit carries the NiFe dinuclear center. Only three Fe/S signals are observed in EPR-monitored reduction by dithionite, NADPH, or internal substrate upon heating. All other clusters presumably have reduction potentials well below that of the H+/H2 couple. Heat-induced reduction by internal substrate allows, for the first time, EPR monitoring of the NiFe center in a hyperthermophilic hydrogenase, which passes through a number of states, some of which are similar to states previously defined for mesophilic hydrogenases. The complexity of the observed transitions reflects a combination of temperature-dependent activation and temperature-dependent reduction potentials.

Mg2+ induces conformational changes in the catalytic subunit of phosphorylase kinase, whether by itself or as part of the holoenzyme complex.

Phosphorylase kinase (PhK) from skeletal muscle is a structurally complex, highly regulated, hexadecameric enzyme of subunit composition (alpha beta gamma delta)4. Previous studies have revealed that the activity of its catalytic gamma subunit is controlled by alterations in quaternary structure initiated at allosteric and covalent modification sites on PhK's three regulatory subunits; however, changes in the conformation of the holoenzyme initiated by the catalytic subunit have been more difficult to document. In this study a monoclonal antibody (mAb gamma79) has been generated against isolated gamma subunit and used as a conformational probe of that subunit. The epitope recognized by this antibody is within the catalytic core of the gamma subunit, between residues 100 and 240, and monovalent fragments of the antibody inhibit the catalytic activity of the holoenzyme, the gamma-calmodulin binary complex, and the free gamma subunit. Activation of PhK by a variety of mechanisms known or thought to act through its regulatory subunits (phosphorylation, ADP binding, or alkaline pH) increased the binding of the holoenzyme to immobilized mAb gamma79, indicating that activation by any of these distinct mechanisms involves repositioning of the portion of the catalytic domain of the gamma subunit containing the epitope for mAb gamma79. The activating ligand Mg2+ also stimulated the binding of the PhK holoenzyme to immobilized mAb gamma79, as well as the binding of mAb gamma79 to immobilized gamma subunit. Thus, Mg2+ increases the accessibility of the mAb gamma79 epitope in both the isolated gamma subunit and in the holoenzyme. Our results suggest that previously reported influences of Mg2+ on the quaternary structure of the PhK holoenzyme are directly mediated by the gamma subunit.

Modulation of recombination human gamma-aminobutyric acidA receptors by isoflurane: influence of the delta subunit.

The gamma-aminobutyric acid (GABA)A receptor/chloride channel has a broad-spectrum anesthetic sensitivity and is a key regulator of arousal. Each receptor/channel complex is an assembly of five protein subunits. Six subunit classes have been identified, each containing one to six members; many combinations are expressed throughout the brain. Benzodiazepines and intravenous anesthetic agents are clearly subunit dependent, but the literature to date suggests that volatile anesthetics are not. The physiological role of the delta subunit remains enigmatic, and it has not been examined as a determinant of anesthetic sensitivity. Combinations of GABA(A) receptor subunit cDNAs were injected into Xenopus laevis oocytes: alpha1beta1, alpha1beta1gamma2L, alpha1beta1delta, and alpha1beta1gamma2Ldelta. Expression of functional ion channels with distinct signalling and pharmacologic properties was demonstrated within 1-4 days by established electrophysiological methods. Co-expression of the delta subunit produced changes in receptor affinity; current density; and the modulatory efficacy of diazepam, zinc, and lanthanum; it also produced subtle changes in the rate of desensitization in response to GABA. Isoflurane enhanced GABA-induced responses from all combinations: alphabeta delta (>10-fold) > alphabeta > alphabeta gamma > or = alphabeta gammadelta (approximately 5-fold). Dose-response plots were bell shaped. Compared with alphabeta gamma receptors (EC50 = 225 microM), both alphabeta delta (EC50 = 372 microM) and alphabeta gammadelta (EC50 = 399 microM) had a reduced affinity for isoflurane. Isoflurane (at a concentration close to the EC50 for each subunit) increased the affinity of GABA for its receptor but depressed the maximal response (alphabeta gamma and alphabeta gammadelta). In contrast, the small currents through alphabeta delta receptors were enhanced, even at saturating agonist concentrations. Delta subunit expression alters GABA(A) receptor function but is not an absolute determinant of anesthetic sensitivity.

TCR gene recombination and alpha beta-gamma delta lineage divergence: productive TCR-beta rearrangement is neither exclusive nor preclusive of gamma delta cell development.

Two types of T lymphocytes can be generated intrathymically, distinguishable by either TCR-gamma delta or -alpha beta surface expression. Regulation of the intrathymic divergence of these cells is unresolved, at least in part because thymically derived gamma delta cells have rarely been studied. We used quantitative Southern blotting together with PCR-based cloning/sequencing and restriction fragment length polymorphism to analyze TCR-alpha and -beta gene recombination in thymically derived gamma delta cells. We found that TCR-beta gene recombination is a frequent occurrence in thymic gamma delta cells. Furthermore, not only do complete (V-D-J) TCR-beta gene rearrangements occur in thymic gamma delta cells, but the frequency of in-frame rearrangements is greater than would be predicted based upon random occurrence. In contrast, we show that thymically derived gamma delta cells do not make detectable rearrangements of the TCR-alpha locus. These studies clearly demarcate a point for alpha beta vs gamma delta commitment in the thymus, after TCR-beta but before TCR-alpha gene recombination. Further, while our data support gamma delta lineage commitment as a consequence of successful TCR-gamma and -delta gene rearrangement, we do not find support for a competitive model of lineage commitment, since productive TCR-beta gene rearrangement does not necessarily relegate cells to the alpha beta lineage.

Melanoma specific Th1 cytotoxic T lymphocyte lines in Vogt-Koyanagi-Harada disease.

To determine the functional properties and cytokine production profiles of melanoma specific cytotoxic T lymphocytes (CTLs) induced from peripheral blood leucocytes of two patients with Vogt-Koyanagi-Harada disease (VKH). Melanoma specific CTL lines were established by long term coculture with a human melanoma cell line (P-36). Cytotoxic activity against P-36 was measured by 51Cr release. The involvement of human leucocyte antigen (HLA) class I or class II molecules in the cytotoxicity of the CTL lines against P-36 was analysed using anti-HLA class I or anti-HLA class II monoclonal antibody (MAb). Surface molecules of CTL lines were analysed by flow cytometry using MAbs specific for CD4, CD8, CD16, CD25, CD56, HLA-DR, T cell antigen receptor (TCR) alpha beta and TCR gamma delta. Cytokine production and soluble interleukin 2 receptor (sIL-2R) secretion were determined by enzyme linked immunosorbent assays. mRNAs of cytokines were analysed using reverse transcription polymerase chain reaction (RT-PCR). CTLs showed strong cytotoxic activity against P-36. The CTL activity of the cell lines against P-36 was inhibited by the anti-HLA-DR MAb, whereas the MAb specific for monomorphic determinants of HLA-A, B, and C failed to block lytic activity. Flow cytometry identified the following surface molecules: CD4+, CD8-, CD16-, CD25+, CD56-, HLA-DR+, TCR alpha beta +, and TCR gamma delta-. CTLs constitutively produced a high level of IL-6. IL-6 production and sIL-2R secretion of CTLs were enhanced when CTLs were stimulated with P-36. CTLs also produced high levels of interferon gamma (IFN-gamma) and IL-2, but not IL-4. mRNAs of IL-2 and IFN-gamma were detected by RT-PCR in the CTLs. Melanoma specific HLADR restricted T helper 1 (Th1) CTLs may play a role in the immunopathogenesis of VKH.

Increased division of alpha beta TCR+ and gamma delta TCR+ intestinal intraepithelial lymphocytes after oral administration of cholera toxin.

Cholera toxin (CT) or its subunits were given orally to mice and division of intestinal intraepithelial lymphocytes (IEL) in vivo measured by double immunofluorescence using 5-bromo-2'-deoxyuridine (BRdU) and membrane alpha beta T-cell receptors (TCR) or gamma delta TCR staining in frozen sections. Cholera toxin (10 micrograms) produced a two- to eightfold-increase in the uptake of BRdU in alpha beta TCR+ IEL in the duodenum and a two-to fivefold increase in gamma delta TCR IEL in the ileum. Increased uptake of BRdU was also seen after a dose of 100 micrograms of CT but this dose was also associated with the loss of alpha beta TCR+ IEL and gamma delta TCR+ IEL in the duodenum. CT-A and CT-B subunit produced increased BRdU incorporation by alpha beta TCR in the duodenum and by gamma delta TCR IEL in the ileum. Cholera toxin therefore appears to be mitogenic for IEL probably due to an indirect mechanism.