Charge exchange recombination spectroscopy at Wendelstein 7-X.

The Charge Exchange Recombination Spectroscopy (CXRS) diagnostic has become a routine diagnostic on almost all major high temperature fusion experimental devices. For the optimized stellarator Wendelstein 7-X (W7-X), a highly flexible and extensive CXRS diagnostic has been built to provide high-resolution local measurements of several important plasma parameters using the recently commissioned neutral beam heating. This paper outlines the design specifics of the W7-X CXRS system and gives examples of the initial results obtained, including typical ion temperature profiles for several common heating scenarios, toroidal flow and radial electric field derived from velocity measurements, beam attenuation via beam emission spectra, and normalized impurity density profiles under some typical plasma conditions.

New advanced operational regime on the W7-AS stellarator.

A promising new plasma operational regime on the Wendelstein stellarator W7-AS has been discovered. It is extant above a threshold density and characterized by flat density profiles, high energy and low impurity confinement times, and edge-localized radiation. Impurity accumulation is avoided. Quasistationary discharges with line-averaged densities n(e) to 4 x 10(20) m(-3), radiation levels to 90%, and partial plasma detachment at the divertor target plates can be simultaneously realized. Energy confinement is up to twice that of a standard scaling. At B(t) = 0.9 T, an average beta value of 3.1% is achieved. The high n(e) values allow demonstration of electron Bernstein wave heating using linear mode conversion.

Genetic comparison of seizure control by norepinephrine and neuropeptide Y.

Epilepsy is a disease of neuronal hyperexcitability, and pharmacological and genetic studies have identified norepinephrine (NE) and neuropeptide Y (NPY) as important endogenous regulators of neuronal excitability. Both transmitters signal through G-protein-coupled receptors, are expressed either together or separately, and are abundant in brain regions implicated in seizure generation. NPY knock-out (NPY KO) and dopamine beta-hydroxylase knock-out (DBH KO) mice that lack NE are susceptible to seizures, and agonists of NE and NPY receptors protect against seizures. To examine the relative contributions of NE and NPY to neuronal excitability, we tested Dbh;Npy double knock-out (DKO) mice for seizure sensitivity. In general, DBH KO mice were much more seizure-sensitive than NPY KO mice and had normal NPY expression, demonstrating that an NPY deficiency did not contribute to the DBH KO seizure phenotype. DKO mice were only slightly more sensitive than DBH KO mice to seizures induced by kainic acid, pentylenetetrazole, or flurothyl, although DKO mice were uniquely prone to handling-induced seizures. NPY contributed to the seizure phenotype of DKO mice at high doses of convulsant agents and advanced stages of seizures. These data suggest that NE is a more potent endogenous anticonvulsant than NPY, and that NPY has the greatest contribution under conditions of extreme neuronal excitability.

Characterization of a phosphoinositide-mediated odor transduction pathway reveals plasma membrane localization of an inositol 1,4, 5-trisphosphate receptor in lobster olfactory receptor neurons.

The role of phosphoinositide signaling in olfactory transduction is still being resolved. Compelling functional evidence for the transduction of odor signals via phosphoinositide pathways in olfactory transduction comes from invertebrate olfactory systems, in particular lobster olfactory receptor neurons. We now provide molecular evidence for two components of the phosphoinositide signaling pathway in lobster olfactory receptor neurons, a G protein alpha subunit of the G(q) family and an inositol 1,4, 5-trisphosphate-gated channel or an inositol 1,4,5-trisphosphate (IP(3)) receptor. Both proteins localize to the site of olfactory transduction, the outer dendrite of the olfactory receptor neurons. Furthermore, the IP(3) receptor localizes to membranes in the ciliary transduction compartment of these cells at both the light microscopic and electron microscopic levels. Given the absence of intracellular organelles in the sub-micron diameter olfactory cilia, this finding indicates that the IP(3) receptor is associated with the plasma membrane and provides the first definitive evidence for plasma membrane localization of an IP(3)R in neurons. The association of the IP(3) receptor with the plasma membrane may be a novel mechanism for regulating intracellular cations in restricted cellular compartments of neurons.

Ethanol-associated behaviors of mice lacking norepinephrine.

Although norepinephrine (NE) has been implicated in animal models of ethanol consumption for many years, the exact nature of its influence is not clear. Lesioning and pharmacological studies examining the role of NE in ethanol consumption have yielded conflicting results. We took a genetic approach to determine the effect of NE depletion on ethanol-mediated behaviors by using dopamine beta-hydroxylase knockout (Dbh -/-) mice that specifically lack the ability to synthesize NE. Dbh -/- males have reduced ethanol preference in a two-bottle choice paradigm and show a delay in extinguishing an ethanol-conditioned taste aversion, suggesting that they drink less ethanol in part because they find its effects more aversive. Both male and female Dbh -/- mice are hypersensitive to the sedative and hypothermic effects of systemic ethanol administration, and the sedation phenotype can be rescued pharmacologically by acute replacement of central NE. Neither the decreased body temperature nor changes in ethanol metabolism can explain the differences in consumption and sedation. These results demonstrate a significant role for NE in modulating ethanol-related behaviors and physiological responses.

Aneuploidy and columnar cuff surveillance after stapled ileal pouch-anal anastomosis in ulcerative colitis.

PURPOSE: A stapled pouch-anal anastomosis without mucosectomy is widely used in restorative proctocolectomy. Uncertainty exists about the longer-term outcome of retaining a columnar cuff of epithelium in the anal canal and about the need for surveillance of the columnar cuff. The aim of this article was to assess the ability to obtain biopsies of the columnar cuff, to assess the risk of dysplasia, and to search for the presence of aneuploidy as an early of marker of dysplasia in nondysplastic epithelium. METHOD: A total of 457 biopsy specimens were taken during 203 examinations of 113 patients. All biopsy specimens were stained with hematoxylin and eosin and examined by microscopy. One hundred thirty-two of these biopsy specimens from 67 patients were frozen and analyzed by flow cytometry for aneuploidy. RESULTS: Mean follow-up after pouch formation was 2.5 years, and the time after diagnosis of ulcerative colitis was 10.1 years. Successful columnar cuff biopsies were done on 93 percent of patients. There was no dysplasia. Two biopsy specimens from one patient had aneuploidy. CONCLUSION: To date, neoplastic change in the columnar cuff is rare. A selective policy of surveillance biopsies is recommended that includes patients greater than ten years after the diagnosis of ulcerative colitis and patients with dysplasia or cancer in their proctocolectomy specimen, but long-term follow-up data are needed.

Norepinephrine-deficient mice have increased susceptibility to seizure-inducing stimuli.

Several lines of evidence suggest that norepinephrine (NE) can modulate seizure activity. However, the experimental methods used in the past cannot exclude the possible role of other neurotransmitters coreleased with NE from noradrenergic terminals. We have assessed the seizure susceptibility of genetically engineered mice that lack NE. Seizure susceptibility was determined in the dopamine beta-hydroxylase null mutant (Dbh -/-) mouse using four different convulsant stimuli: 2,2,2-trifluroethyl ether (flurothyl), pentylenetetrazol (PTZ), kainic acid, and high-decibel sound. Dbh -/- mice demonstrated enhanced susceptibility (i.e., lower threshold) compared with littermate heterozygous (Dbh +/-) controls to flurothyl, PTZ, kainic acid, and audiogenic seizures and enhanced sensitivity (i.e., seizure severity and mortality) to flurothyl, PTZ, and kainic acid. c-Fos mRNA expression in the cortex, hippocampus (CA1 and CA3), and amygdala was increased in Dbh -/- mice in association with flurothyl-induced seizures. Enhanced seizure susceptibility to flurothyl and increased seizure-induced c-fos mRNA expression were reversed by pretreatment with L-threo-3, 4-dihydroxyphenylserine, which partially restores the NE content in Dbh -/- mice. These genetically engineered mice confirm unambiguously the potent effects of the noradrenergic system in modulating epileptogenicity and illustrate the unique opportunity offered by Dbh -/- mice for elucidating the pathways through which NE can regulate seizure activity.

Cross-species reactivity of a panel of antibodies with monkey and porcine tissue.

The continuing shortage of organs available for transplantation limits the number of patients able to benefit from this highly successful form of therapy. Interest in alternative sources of organs has now turned towards the pig because of its physiological similarity to human. There is a requirement therefore for reagents not only for research purposes but possibly for studying xenotransplants in the clinical situation in the future. In this study, we have concentrated on determining the cross-species reactivity of a large panel of antibodies directed against human leukocyte markers, testing peripheral blood leukocytes and also including renal tissue to determine non-leukocyte cross-reactivity. A total of 63 out of 127 antibodies cross-reacted with cynomolgus monkey cells. Twenty of these antibodies stained similar populations of leukocytes to human, whereas the remaining 43 reacted with clearly different populations. The majority of antibodies (108/127) were unreactive with porcine leukocytes, reflecting the evolutionary differences between pig and man. Of the 19 antibodies cross-reactive with porcine cells, seven reacted with similar proportions of leukocytes to human, whereas the remaining 12 antibodies stained entirely different populations. The most interesting, and potentially most useful, antibodies were four that reacted with human, cynomolgus monkey and porcine tissue in a similar manner, suggesting that the epitopes recognized are present on similar molecules. These antibodies were directed against CD29 (MEM1O1A, K20) and CD18 (BU87, 7E4), the common beta1- and beta2-integrin subunits respectively. This study demonstrates that there are antigens common to cynomolgus monkey, pig and man that react with currently available antibodies. Nevertheless, when determining cross-species reactivity of human antibodies, it is important to consider the possibility that there may be additional non-leukocyte reactivity in other tissues.

Cutting edge: rapid cloning of tumor-specific CTL suitable for adoptive immunotherapy of melanoma.

Adoptive immunotherapy using CTL has provided some clinical benefit to patients with metastatic melanoma. Use of cloned CTL of known specificity might improve clinical effect, but technical difficulties have limited exploration of this possibility. We have used fluorescence-driven cell sorting to clone tumor-specific CTL after staining with tetrameric MHC class I/peptide complexes. CTL specific for the melanoma Ags melan-A, tyrosinase, and MAGE3 were cloned from the peripheral blood, tumor-infiltrated lymph nodes, and skin metastases of five patients. Clones were isolated and characterized in as little as 6 weeks, much faster than is possible with previous techniques. We show that these CTL clones express markers compatible with immunotherapeutic use in melanoma, including the cutaneous lymphocyte Ag, which is associated with homing to skin.

Independent contributions of HLA epitopes and killer inhibitory receptor expression to the functional alloreactive specificity of natural killer cells.

Human NK cells express receptors (KIR) which inhibit lysis through binding to HLA class I on target cells. KIR expression in different individuals has not been intensively investigated and it is not known how the KIR repertoire relates to HLA type or influences the overall activity of NK populations. This may be important in the response of NK cells to HLA-mismatched organ transplants since the ligands for KIR are supertypic epitopes shared between certain HLA alleles. We studied the effect of matching for HLA on the cytotoxicity of NK cells from individuals homozygous or heterozygous for relevant HLA class I epitopes and correlated this with KIR expression and genotype. Considerable variation in the KIR repertoire of different donors was evident, including functional KIR expressed in the absence of specific HLA ligands. We confirmed the predominant influence of HLA-C in a hierarchy of inhibitory effects mediated by HLA class I loci. In certain individuals, inhibition patterns are more complicated and may be due to the relative expression of the CD94/NKG2 receptors. Our study reveals the separate contributions of HLA and KIR molecules to NK cell alloreactivity and provides a basis for consideration of the functional diversity of KIR genes in transplantation.

Immunoregulation by CD4 T cells in the induction of specific immunological unresponsiveness to alloantigens in vivo: evidence for a reduction in the frequency of alloantigen-specific cytotoxic T cells in vitro.

Donor-specific unresponsiveness to allogeneic cardiac allografts in mice can be induced by the combined pretreatment with donor alloantigen and anti-CD4 antibody (anti-CD4+DST). We have investigated whether the induction of unresponsiveness in this model is due to the presence of T cells that regulate immune responsiveness towards the allograft. First, we analysed the functional characteristics of splenocytes from pretreated mice at the time of transplantation. A significant reduction in the frequency of donor specific cytotoxic precursor was found only after the anti-CD4+DST treatment. Next, we designed an in vitro assay to identify the phenotype of the splenocyte population responsible. CD4+ and CD4- fractions were purified from mice treated with anti-CD4+DST or anti-CD4 alone (controls) by cell sorting. Interestingly, only the addition of CD4+ cells from anti-CD4+DST treated mice resulted in a selective reduction and a bimodal distribution in the donor specific CTLp response, indicating the presence of a regulatory population. CD4+ cells from controls did not have this effect. These in vitro findings were substantiated by adoptive transfer experiments in vivo. These data demonstrate that CD4+ cells with the ability to regulate immune responsiveness to a cardiac allograft are present at the time of transplantation following pretreatment with donor alloantigen in combination with anti-CD4.

Direct isolation, phenotyping and cloning of low-frequency antigen-specific cytotoxic T lymphocytes from peripheral blood.

Cytotoxic T lymphocytes (CTLs) play an important role in controlling viral infections and certain tumours, but characterising specific CTL responses has always been technically limited. Fluorogenic 'tetramers' of major histocompatibility complex (MHC) class I complexes have been exploited recently to quantify the massive expansion of specific CTLs in human immunodeficiency virus (HIV) infection [1]. Here, we use MHC class I complex tetramers to isolate low-frequency antigen-specific CTLs directly from human peripheral blood, allowing the simultaneous phenotypic and functional characterisation and cloning of these CTLs. We synthesised a tetramer that specifically stained human leukocyte antigen (HLA)-A2. 1-restricted CTL clones recognising the influenza matrix protein peptide 58-66, matrix 58-66 [2]. This tetramer stained between 1 in 1,500 and 1 in 58,000 peripheral blood mononuclear cells (PBMCs) from HLA-A2.1+ individuals. The surface phenotype of these cells could be analysed by fluorescence-activated cell sorting (FACS), and the cells could be directly sorted into enzyme-linked immunospot (ELISpot) plates, where they released interferon-gamma (IFN-gamma) within 1 day of antigen exposure. The same population was cloned by FACS, and the specificity of several expanded clones was confirmed. Cloning was greatly simplified and accelerated compared with standard protocols, and was highly efficient. We also used tetramer-based sorting to enrich melanoma-specific CTLs derived from a tumour-infiltrated lymph node. Direct cloning of specific CTLs from peripheral blood can provide important information about immunological memory, CTL responses against tumour antigens and CTL proliferation and function, and opens up new possibilities for generating CTLs for adoptive immunotherapy.

Functional and biochemical subtypes of the haplotype HLA-DR3 in patients with celiac disease or idiopathic membranous nephropathy.

HLA class II beta-chain polymorphism was investigated in the haplotype HLA-DR3 to determine if patients with HLA-DR3-associated diseases express normal or variant class II polymorphisms. Analysis was carried out by two-dimensional gel electrophoresis of immunoprecipitated HLA class II molecules, DNA hybridization with DR beta and DQ beta gene probes on Taq 1, Bam H1, or Rsa 1 digests, and mixed lymphocyte culture. Two subtypes of HLA-DR3 were identified in normal homozygous DR3 individuals on the basis of polymorphism in one of two DR beta chains detected, corresponding to differences in DR beta restriction fragment patterns. These polymorphisms exhibited significant linkage disequilibrium with the A1,B8,DR3 and B18,DR3 haplotypes, respectively. In proliferative experiments, cells with the B18,DR3-associated polymorphism strongly stimulated cells from donors with the B8,DR3-related polymorphism, suggesting that a T-cell epitope recognized by B8,DR3 cells lies on the B18,DR3-associated DR beta chain. In seven HLA-DR3 homozygous patients with celiac disease and three HLA-DR3-homozygous patients with idiopathic membranous nephropathy, only the normal patterns of HLA class II molecules were displayed, the B8,DR3 type occurring in all patients and the B18,DR3 type in one patient. These data suggest that celiac disease and idiopathic membranous nephropathy are not related to disease-specific HLA-DR beta or -DQ beta gene variants within the DR3 population that are revealed by these methods.

HLA-DR2 subtypes form an additional supertypic family of DR beta alleles.

Homozygous lymphoblastoid cell lines representing Dw subtypes of the DR2 serotype were studied for structural polymorphism at DR beta. These subtypes included Dw2, Dw12, and non-Dw2/non-Dw12. Analysis by two-dimensional gel electrophoresis showed that two DR beta genes were expressed in each cell line studied. One of these expressed genes encoded a protein that was nonpolymorphic on two-dimensional gel electrophoresis among all subtypes. The second expressed DR beta gene was polymorphic and migrated to a position on two-dimensional gels dependent on the subtype of the cell line. cDNA sequence analysis of the DR beta genes revealed a DR beta gene identical between the Dw2 and Dw12 subtypes, which correlates with the nonpolymorphic spot on two-dimensional gels. A second gene was sequenced that exhibited variability between the Dw2 and Dw12 subtypes. This variability takes the form of clustered point mutations in the first domain of the molecules. The alleles from the DR beta genes of a non-Dw2/non-Dw12 cell line, AZH, were unusual in sequence. In contrast to other DR beta alleles, the AZH genes may have been generated by a double recombinational event between two DR beta loci from a DR2 parent. The DR2 serotype may also constitute a supertypic "family," with one DR beta gene relatively nonpolymorphic, and one that varies with Dw subtype.

Structural and functional variability among DQ beta alleles of DR2 subtypes.

Homozygous lymphoblastoid cell lines representing various Dw subtypes of DR2 were examined for polymorphism at the DQ beta locus by molecular and cellular techniques. The subtypes studied included Dw2, Dw12, and a group heterogenous by cellular typing that we shall refer to as non-Dw2/non-Dw12. Restriction fragment length polymorphism analysis of cell lines representing these subtypes revealed DQ beta-specific patterns consistent with cellular typing. Two-dimensional gel electrophoresis of DQ molecules from representative cell lines revealed a structural polymorphism of DQ beta among the three subtypes. The DQ beta chain migrated to a position that was unique to each subtype and was consistent among various representative cell lines of each subtype. Nucleotide sequence analysis of cDNA clones of DQ beta from Dw2, Dw12, and non-Dw2/non-Dw12 lines confirmed that the variability resided at the genetic level. Variability was found in the form of numerous scattered nucleotide substitutions throughout the first domain of these alleles. The DQ beta gene of the non-Dw2/non-Dw12 cell line AZH was further found to be almost identical with the DQ beta gene of a DR1 line (Bell et al. 1985b), implicating a common evolutionary origin of these alleles. The only difference between these two sequences was due to an apparent gene conversion event at amino acid 57. T-cell cloning experiments resulted in the derivation of Epstein-Barr virus-specific, DQw1-restricted clones that proliferated against only those cell lines that exhibited the DQ beta gene common to AZH and the DR1 cell line. Thus, the polymorphism among DQ beta alleles within DR2 results in subtype-specific restriction.

Simplification of two-dimensional gel patterns of HLA class II antigens.

Two-dimensional gel electrophoresis of material immunoprecipitated from radiolabeled cells by anti-HLA class II monoclonal antibodies has been a useful technique but the patterns observed have varied considerably between laboratories. In this study we investigated the parameters that vary between different laboratories. We found that heterogeneity due to post synthetic modification of beta and alpha chains of DR and DQ can be avoided by labeling cells for 30 min with 35S-methionine. A mixture of ampholytes pH range 3.5-10:5-7 of 1:4 run for 3000 V/h gave the best resolution of beta and alpha chains. With this technique we could clearly reveal differences in the DR beta-2 chains of those DR2 haplotypes, Dw2, Dw12 and LD'tb24. The Dr beta 1 position varied between different DR types.

Monoclonal antibody to human platelet glycoprotein I. I. Immunological studies.

A monoclonal hybridoma antibody specific for platelet glycoprotein I complex is described. The nature od the antigen was determined by demonstration that it was chymotrypsin sensitive and gave a peak at 150 000 daltons on SDS-PAGE after immunoprecipitation. The expression of the antigen is restricted to platelets and megakaryocytes with at least 1.6 x 10(4) molecules of antigen per platelet. The antibody failed to bind to platelets from patients with Bernard Soulier syndrome, where there is known to be a deficiency of glycoprotein Ib/Is expression. Binding to platelets from patients with Glanzmann's thrombasthenia was normal.

A monoclonal antibody that recognizes an antigenic determinant shared by HLA A2 and B17.

A hybridoma monoclonal anti-HLA antibody has been produced by the technique of Kohler and Milstein [1]. This antibody recognizes a new specificity common to HLA A2 and B17. It was shown to be a single antibody by isoelectric focusing and absorption experiments.