Vaccine-induced allo-HLA-reactive memory T cells in a kidney transplantation candidate.

BACKGROUND: Allo-human leukocyte antigen (HLA) reactivity by naturally acquired viral-specific memory T cells is common. However, the effect of successful vaccination on the alloreactive memory T-cell repertoire is unclear. We hypothesized that vaccination could specifically induce allo-HLA-reactive memory T cells. METHODS: A varicella-zoster virus (VZV) immediate early 62 (IE62)-specific CD8 memory T-cell clone was single cell sorted from a VZV seronegative renal transplant candidate after response to live attenuated varicella vaccination. To analyze the allo-HLA reactivity, the VZV IE62-specific T-cell clone was tested against HLA-typed target cells and target cells transfected with HLA molecules, in both cytokine production and cytotoxicity assays. RESULTS: The varicella vaccine-induced VZV IE62-specific T-cell clone specifically produced interferon-γ when stimulated with HLA-B*55:01-expressing Epstein-Barr virus-transformed B cells and HLA-B*55:01-transfected K562 cells (single HLA antigen expressing cell line [SALs]) only. The clone also demonstrated specific cytolytic effector function against HLA-B*55:01 SALs and phytohemagglutinin blasts. Cytotoxicity assays using proximal tubular epithelial cell and human umbilical vein endothelial cell targets confirmed the kidney tissue specificity of the allo-HLA-B*55:01 reactivity, and the relevance of the cross-reactivity to clinical kidney transplantation. The results also suggest that molecular mimicry, and not bystander proliferation, is the mechanism underlying vaccine-induced alloreactivity. CONCLUSIONS: Varicella vaccination generated a de novo alloreactive kidney cell-specific cytolytic effector memory T cell in a patient awaiting renal transplantation. Vaccination-induced alloreactivity may have important clinical implications, especially for vaccine timing and recipient monitoring.

Tissue specificity of cross-reactive allogeneic responses by EBV EBNA3A-specific memory T cells.

BACKGROUND: The crossreactivity of Epstein-Barr virus (EBV Epstein-Barr virus nuclear antigen 3A [EBNA3A])-specific CD8 T cells against allogeneic human leukocyte antigen (HLA)-B*44:02 has been shown to be dependent on presentation of self-peptide EEYLQAFTY by the target antigen. In this study, we report that allogeneic HLA-B*44:02 proximal tubular epithelial cells (PTECs) and human umbilical vein endothelial cells (HUVECs) are poor targets for EBV EBNA3A-specific T cells. METHODS: The EEY peptide was exogenously loaded onto HLA-B*44:02 and HLA-B*44:03-expressing PTECs and HUVECs. EEY-peptide-loaded, and unloaded, PTECs and HUVECs were then incubated with serial dilutions of our EBNA3A T-cell clone, in a cytotoxicity assay. RESULTS: Although HLA-B*44:02-expressing PTECs were specifically lysed in proportion to the effector/target ratio by the EBNA3A T-cell clone, without peptide loading, lysis was greatly increased by exogenous EEY peptide loading (15% vs. 75%; P<0.0001). HLA-B*44:02-expressing HUVECs were only lysed when loaded with exogenous EEY peptide (0% vs. 64%; P<0.0001). Lack of HLA expression and lack of ABCD3 gene expression were excluded as a cause for these results. PTECs and HUVECs were specifically targeted by another alloreactive T-cell clone without exogenous peptide loading, suggesting that the lack of recognition of HLA-B*44:02 epithelial and endothelial cells by the EBV EBNA3A T-cell clone was due to lack of EEYLQAFTY peptide presentation. CONCLUSIONS: Tissue-specific (peptide dependent) alloreactivity may have important implications for transplantation monitoring and rejection.

Allo-HLA reactivity of virus-specific memory T cells is common.

Graft-versus-host disease and graft rejection are major complications of allogeneic HLA-mismatched stem cell transplantation or organ transplantation that are caused by alloreactive T cells. Because a range of acute viral infections have been linked to initiating these complications, we hypothesized that the cross-reactive potential of virus-specific memory T cells to allogeneic (allo) HLA molecules may be able to mediate these complications. To analyze the allo-HLA reactivity, T cells specific for Epstein-Barr virus, cytomegalovirus, varicella zoster virus, and influenza virus were tested against a panel of HLA-typed target cells, and target cells transduced with single HLA molecules. Eighty percent of T-cell lines and 45% of virus-specific T-cell clones were shown to cross-react against allo-HLA molecules. The cross-reactivity of the CD8 and CD4 T-cell clones was directed primarily against HLA class I and II, respectively. However, a restricted number of CD8 T cells exhibited cross-reactivity to HLA class II. T-cell receptor (TCR) gene transfer confirmed that allo-HLA reactivity and virus specificity were mediated via the same TCR. These results demonstrate that a substantial proportion of virus-specific T cells exert allo-HLA reactivity, which may have important clinical implications in transplantation settings as well as adoptive transfer of third-party virus-specific T cells.

The human gamma-globin TATA and CACCC elements have key, distinct roles in suppressing beta-globin gene expression in embryonic/fetal development.

The competition model of globin gene regulation states that the gamma-globin gene precludes expression of the beta-globin gene in early development by competing for the enhancing activity of the locus control region. The gamma-globin gene with a -161 promoter is sufficient for suppressing beta-globin gene expression, and the gamma-globin TATA and CACCC elements are necessary for this effect. In this work, stable transfection and transgenic mouse assays have been performed with constructs containing HS3 and HS2 from the locus control region, the gamma-globin gene with promoter mutation(s), and the beta-globin gene. The data indicate that the gamma-globin TATA and CACCC elements together have at least an additive effect on the beta/gamma-globin mRNA ratio in early erythroid cells, suggesting that the elements work coordinately to suppress beta-globin gene expression. The TATA and CACCC are the major gamma-globin promoter elements responsible for this effect. Transgenic mouse experiments indicate that the gamma-globin TATA element plays a role in gamma-globin expression and beta-globin suppression in the embryo and fetus; in contrast, the CACCC element has a stage-specific effect in the fetus. The results suggest that, as is true for the erythroid Krüppel-like factor (EKLF) and the beta-globin promoter CACCC, a protein(s) binds to the gamma-globin CACCC element to coordinate stage-specific gene expression.

Human RAD52 exhibits two modes of self-association.

The human RAD52 protein plays an important role in the earliest stages of chromosomal double-strand break repair via the homologous recombination pathway. Individual subunits of RAD52 self-associate into rings that can then form higher order complexes. RAD52 binds to double-strand DNA ends, and recent studies suggest that the higher order self-association of the rings promotes DNA end-joining. Earlier studies defined the self-association domain of RAD52 to a unique region in the N-terminal half of the protein. Here we show that there are in fact two experimentally separable self-association domains in RAD52. The N-terminal self-association domain mediates the assembly of monomers into rings, and the previously unidentified domain in the C-terminal half of the protein mediates higher order self-association of the rings.

Factors that influence the decision to do an adequate evaluation of a patient with a positive stool for occult blood.

OBJECTIVE: The factors that influence the decision to do an adequate evaluation for a positive test for fecal occult blood in a middle-aged or elderly patient are largely unknown. Our study was undertaken to determine whether factors such as the number of positive Hemoccult II card windows, age, gender, family history of colon cancer, the patient's concern that he or she might have colon cancer, or history of rectal bleeding influence the evaluation performed. METHOD: A mass screening program for colon cancer was performed using unrehydrated Hemoccult II cards in the Boston area. RESULTS: Among the 23,593 Hemoccult II cards returned to Beth Israel Deaconess Medical Center, cards from 1,112 patients (4.7%) were found to be positive for one or more of the six possible card windows. Ninety percent, or 940 patients, over 40 yr of age had follow-up information available. As the number of positive windows increased from one to four, there was a significant trend (p < 0.001) for the adequacy of the evaluation to increase. Family history (p = 0.044) and a patient's worry that he or she might have colon cancer (p = 0.003) significantly improved a patient's chance for an adequate evaluation. CONCLUSIONS: Hemoccult testing is not followed by an adequate evaluation in a significant proportion of patients. Our study points out for the first time that the number of positive Hemoccult windows significantly influences the decision-making.

Acute tryptophan depletion in schizophrenia.

BACKGROUND: Brain 5-hydroxytryptamine (5-HT) function is implicated in the pathophysiology of schizophrenia and the action of new generation antipsychotic drugs. By the method of acute tryptophan depletion (ATD) 5-HT can be selectively manipulated. The aim of this study was to examine the effects of ATD on symptoms, mood and cognition in schizophrenic patients. METHODS: Twenty-eight schizophrenic patients participated in a within subject, double-blind, placebo-controlled counterbalanced cross-over study. Patients with a concurrent DSM-IV axis I diagnosis were excluded. Symptoms, mood and cognitive function were evaluated following ATD or ingestion of a control drink. RESULTS: The depleting drink significantly reduced plasma total and free tryptophan. Tryptophan/LNAA ratios did not alter with the administration of the control drink, but differed significantly with ATD; however there was no significant change in tyrosine/LNAA ratio. ATD led to impairment in executive function that was dependent upon the order of administration. Tests of sustained attention, speed of processing, and everyday memory were not affected. No effects were observed on subjective mood ratings, movement disorders or PANSS scores. CONCLUSIONS: Acute tryptophan depletion selectively alters cognition in schizophrenia, but has no effect on symptoms, mood ratings or movement disorders.

Fenbendazole treatment without environmental decontamination eradicates Syphacia muris from all rats in a large, complex research institution.

Syphacia muris parasitism was eliminated from rats and voles by feeding fenbendazole-medicated chow (150 ppm) for five 7-day periods; treatment periods were separated by 7-day periods of feeding non-medicated chow, yielding atotal treatment course of 9 weeks. No other manipulations to facilitate eradication, including the use of filter tops, autoclaved cages, environmental decontamination, colony depopulation, breeding cessation, and research restriction, were done. The examination of 3143 cellophane-tape impressions of the anus and 160 cecal examinations from euthanized rats and voles during the treatment period and for 7 months afterwards confirmed the efficacy of treatment. Treatment was rapidly effective in voles. In rats, pinworm eggs persisted at high levels for 2 weeks after the start of treatment, but no eggs were found after 22 days.

The gammaPE complex contains both SATB1 and HOXB2 and has positive and negative roles in human gamma-globin gene regulation.

A large nuclear protein complex, termed gammaPE (for gamma-globin promoter and enhancer binding factor), binds to five sites located 5' and 3' of the human y-globin gene. Two proteins, SATB1 (special A-T-rich binding protein 1) and HOXB2, can bind to yPE binding sites. SATB1 binds to nuclear matrix-attachment sites, and HOXB2 is a homeodomain protein important in neural development that is also expressed during erythropoiesis. The present work showed that antisera directed against either SATB1 or HOXB2 reacted specifically with the entire gammaPE complex in electrophoretic mobility shift assays (EMSAs), suggesting that the two proteins can bind to the gammaPE binding site simultaneously. When SATB1 or HOXB2 was expressed in vitro, they could bind independently to gammaPE binding sites in EMSA. Interestingly, the proteins expressed in vitro competed effectively with each other for the gammaPE binding site, suggesting that this may occur under certain conditions in vivo. Transient cotransfections of a HOXB2 cDNA and a y-globin-luciferase reporter gene construct into cells expressing SATB1 suggested that SATB1 has a positive and HOXB2 a negative regulatory effect on transcription. Taking into account their potentially opposing effects and binding activities, SATB1 and HOXB2 may modulate the amount of gamma-globin mRNA expressed during development and differentiation.

A combination of hydroxyurea and isobutyramide to induce fetal hemoglobin in transgenic mice is more hematotoxic than the individual agents.

Pharmacologic agents such as hydroxyurea (HU), N, 3-4 trihydroxybenzamide (didox), and isobutyramide (ISB) can elevate gamma-globin as a potential treatment for the beta-hemoglobinopathies. In these experiments, transgenic mice with 5'HS2 from the human beta-globin locus control region, the fetal (A gamma), and adult (beta s) globin genes were used. Mice were treated with HU, didox, or ISB individually, or with combinations of HU or didox with ISB. The aim was to determine whether these drugs have synergistic effects on the induction of fetal hemoglobin (HbF) and whether the combination regimens are more hematotoxic. In the combination regimens, injections of HU or didox for five weeks were concomitant with ISB treatment every other day for the final three weeks of treatment. The combination of HU + ISB was more hematotoxic than the individual drugs based on significantly increased percentages of reticulocytes and reduced hemoglobin, indicating that caution should be taken in treatments involving combinations of these types of drugs. The didox + ISB combination was not more hematotoxic than the individual drugs. HbF was not induced in the groups treated with the combinations of HU or didox with ISB compared to the individual agents. There was a negligible effect on the percentage of HbF and an unexpected negative effect on the percentage of F cells. The results also have implications for future testing of HbF-inducing drugs in mouse models. In control mice that were phlebotomized but not treated with any drugs, increased percentages of F cells were observed, indicating that blood sampling can cause this effect. In addition, increases in the percentage of F cells did not correlate with increases in the percentage of HbF, indicating that monitoring F cells alone is not a sufficient measure of HbF induction.

The gamma-globin promoter has a major role in competitive inhibition of beta-globin gene expression in early erythroid development.

The human gamma-globin gene competitively inhibits beta-globin gene expression in early erythroid development. To identify the gamma-globin gene sequences required for this effect, transgenic mice and stable transfection analyses with constructs containing 5'HS2 from the locus control region, modified gamma-globin genes, and the beta-globin gene were used. The -136 to +56 region of the gamma-globin promoter is necessary for competitive inhibition, as the beta-globin gene was inappropriately expressed in mouse embryos and in K562 and HEL cells containing constructs in which this region was deleted. Independently, the -140 to +56 region of gamma-globin gene was not sufficient to inhibit beta-globin transcription in mouse embryos or in cultured cells. Competitive inhibition of beta-globin gene expression was observed in K562 and HEL cells having a gamma-globin gene with a -161 promoter. The data suggest that the -161 gamma-globin promoter, which includes the CACCC box, two CCAAT boxes, the stage selector element (SSE), and TATA box, has a major role in suppressing beta-globin transcription early in development. Proteins binding to these or other gamma-globin promoter elements may interact with those binding to the locus control region, consequently precluding beta-globin transcription.

The roles of 5'-HS2, 5'-HS3, and the gamma-globin TATA, CACCC, and stage selector elements in suppression of beta-globin expression in early development.

The roles of HS2 and HS3 from the human beta-globin locus control region and of the TATA, CACCC, and stage selector elements of the gamma-globin promoter, in competitive inhibition of beta-globin gene expression in early development, were tested using stable transfections of HEL and K562 cells. Cells with an HS3gamma beta construct demonstrate that HS3 exhibits enhancing activity, but compared with HS2, this site participates less consistently in the inhibition of embryonic/fetal beta-globin expression. In cells with HS3HS2gamma beta constructs, the two HS sites act in concert to more effectively enhance gamma-globin gene expression and to drive stage-specific expression of the gamma- and beta-globin genes. A gamma-globin gene with a -161 promoter can competitively inhibit beta-globin gene expression. HS3HS2gamma beta constructs were used to determine the effects of gamma-globin promoter mutations within this region on competition. The CACCC and TATA elements, but not the stage selector element, inhibit inappropriate embryonic/fetal stage expression of the beta-globin gene. The mutation in the gamma-globin TATA element results in the use of two major alternative transcription start sites. The data suggest that proteins binding to the gamma-globin CACCC and TATA elements interact with those binding to HS2 and/or HS3 to preclude beta-globin transcription in early development.

Positive transcriptional regulation of the human gamma-globin gene. Gamma PE is a novel nuclear factor with multiple binding sites near the gene.

A novel nuclear factor involved in human gamma-globin gene regulation has been identified. Co-migrating and cross-competing complexes were formed with five individual fragments from the 5'- and 3'-flanking regions of the gene in DNA-protein binding assays. This indicates that a nuclear factor, termed gamma PE, has multiple binding sites near the gamma-globin gene. This characteristic is shared by other important factors in globin gene regulation, such as GATA-1. The five gamma PE binding sites can be placed in two categories based on DNA-protein binding affinity and DNA sequence composition. The consensus sequence for the two higher affinity binding sites is ATTANNNGGAANNCT(N)TNNNTAATGG and for the three lower affinity sites is AAAAN(A/T)A(A/T)TT. Both the ATTA and the TAAT motifs of a high affinity binding site are required for efficient DNA-protein binding. The tissue distribution of gamma PE binding activity is broad, including both erythroid and non-erythroid cell types. Transcription of either a gamma-globin or heterologous promoter is increased in the presence of nearby gamma PE binding sites. Therefore, gamma PE may be involved in activating the gamma-globin gene in fetal erythroid cells. UV cross-linking analysis indicates that the major protein interacting with a high affinity gamma PE binding site has a molecular mass of 108 kDa.

Regulated expression of the human beta globin gene in transgenic mice requires an upstream globin or nonglobin promoter.

Transgenic mice have been used extensively to study elements governing the erythroid-specific developmental switch from human fetal gamma to human adult beta globin. Previous work demonstrated that a small construct composed of hypersensitive site 2 (HS2) of the locus control region (LCR) linked to the gamma and beta globin genes (HS2-gamma-beta) is sufficient for correct tissue and temporal expression of these genes, whereas HS2-beta alone is inappropriately expressed in the embryo. Two models, which are not mutually exclusive, have been proposed to explain these results and those of other constructs in transgenic mice. One model emphasizes the conserved polarity in the globin locus and suggests a distance effect whereby the beta globin gene must be removed from the LCR/HS2 to prevent an early and incorrect activation of this gene in the embryonic compartment. A second hypothesis proposes a competition between the gamma and beta globin gene promoters for interaction with the LCR/HS2. The active gamma globin gene promoter positioned between the LCR/HS2 and the beta globin gene thereby interacts with the HS2 elements early in erythroid development and is expressed until a change in putative stage-specific nuclear factors makes an interaction with the adult beta globin gene more favorable. In an effort to test the competition model, a construct has been prepared in which a small deletion was produced in the promoter region of the gamma globin gene while in the context of the HS2-gamma-beta plasmid. Analysis of this construct in transgenic mice reveals a constitutive unregulated expression of the human beta globin gene during erythroid development. To determine if this competition effect is specific for globin genes, a heterologous reporter gene has been substituted for the gamma globin gene in the construct HS2-gamma-beta. In this case, the beta globin gene exhibits correct developmental expression. This data is consistent with a model in which transcription from a promoter upstream of the beta globin gene in some manner protects this adult gene from activation by the LCR/HS2 during early development.

Human gamma- to beta-globin gene switching using a mini construct in transgenic mice.

The developmental regulation of the human globin genes involves a key switch from fetal (gamma-) to adult (beta-) globin gene expression. It is possible to study the mechanism of this switch by expressing the human globin genes in transgenic mice. Previous work has shown that high-level expression of the human globin genes in transgenic mice requires the presence of the locus control region (LCR) upstream of the genes in the beta-globin locus. High-level, correct developmental regulation of beta-globin gene expression in transgenic mice has previously been accomplished only in 30- to 40-kb genomic constructs containing the LCR and multiple genes from the locus. This suggests that either competition for LCR sequences by other globin genes or the presence of intergenic sequences from the beta-globin locus is required to silence the beta-globin gene in embryonic life. The results presented here clearly show that the presence of the gamma-globin gene (3.3 kb) alone is sufficient to down-regulate the beta-globin gene in embryonic transgenic mice made with an LCR-gamma-beta-globin mini construct. The results also show that the gamma-globin gene is down-regulated in adult mice from most transgenic lines made with LCR-gamma-globin constructs not including the beta-globin gene, i.e., that the gamma-globin gene can be autonomously regulated. Evidence presented here suggests that a region 3' of the gamma-globin gene may be important for down-regulation in the adult. The 5'HS2 gamma en beta construct described is a suitable model for further study of the mechanism of human gamma- to beta-globin gene switching in transgenic mice.

Transcription factor OTF-1 interacts with two distinct DNA elements in the A gamma-globin gene promoter.

A DNA region (site II) in the promoter of the human A gamma-globin gene (-182 to -168) is involved in transcriptional regulation. At least two nuclear proteins bind to this region: the erythroid-specific factor NF-E1/GF-1 and another factor present in many cell lines. In the present study, we demonstrate that the ubiquitous factor binding to site II has immunological identity with the octamer transcription factor OTF-1, which has been implicated in the regulation of expression of genes such as histone H2b and small nuclear RNA. In addition, we show that OTF-1 binds to site I (-291 to -267), a purine-rich region upstream of site II. Interestingly, OTF-1 binds to sites I and II with equal affinity. This was unexpected since the 14 bp site I binding site AAGAATAAATTAGA (-291 to -278), determined by methylation interference, does not show obvious similarities to the canonical octamer binding site for OTF-1 in site II (ATGCAAAT). Interaction of OTF-1 with functionally active binding sites in the gamma-globin promoter suggests that this factor has a role in gamma-globin transcription.

Mutations in two regions upstream of the A gamma globin gene canonical promoter affect gene expression.

Two regions upstream of the human fetal (A gamma) globin gene, which interact with protein factors from K562 and HeLa nuclear extracts, have functional significance in gene expression. One binding site (site I) is at a position -290 to -267 bp upstream of the transcription initiation site, the other (site II) is at -182 to -168 bp. Site II includes the octamer sequence (ATGCAAAT) found in an immunoglobulin enhancer and the histone H2b gene promoter. A point mutation (T----C) at -175, within the octamer sequence, is characteristic of a naturally occurring HPFH (hereditary persistence of fetal hemoglobin), and decreases factor binding to an oligonucleotide containing the octamer motif. Expression assays using a A gamma globin promoter-CAT (chloramphenicol acetyl transferase) fusion gene show that the point mutation at -175 increases expression in erythroid, but not non-erythroid cells when compared to a wild-type construct. This correlates with the actual effect of the HPFH mutation in humans. This higher expression may result from a mechanism more complex than reduced binding of a negative regulator. A site I clustered-base substitution gives gamma-CAT activity well below wild-type, suggesting that this factor is a positive regulator.

Sites I and II upstream of the A gamma globin gene bind nuclear factors and affect gene expression.

Five broad regions in and near the A gamma globin gene specifically bind proteins in K562 nuclear extracts. Two are located 5' of the gene, one between -1349 and -1094, and the other between -384 and +52. Each of the two introns has a binding region, +184 to +284 and +930 to +1209. The fifth binding region is within the A gamma globin gene enhancer, a 750 bp Hind III fragment located 3' to the gene. The nuclear factors which bind to any or all of these regions may be important for control of regulation of the A gamma globin gene. Since HPFH point mutations occur at -175, -196 and -202, we investigated K562 nuclear factor binding to this region more closely. Footprints were obtained for two binding sites, one from -168 to -185 (site II) and the other from -267 to -293 (site I). Site II contains an octamer sequence (ATGCAAAT) important for protein binding and expression of the histone H2b and immunoglobulin genes. The HPFH mutation at -175 changes the T in the octamer sequence to a C. Site I is upstream of the bases affected by HPFH mutations. A A gamma globin gene promoter - CAT reporter fusion gene was constructed with a clustered - base substitution of site I or a T----C point mutation at -175. The mutation in site I decreases CAT expression 20X compared to wild-type in erythroid and non-erythroid cells. Site I probably binds a positive regulator. The T----C change at -175 increases expression 2-3X over wild-type in erythroid cells, but not in non-erythroid cells. This increase correlates with the effect of the naturally occurring HPFH, and may result from decreased binding of a negative effector, and/or increased positive factor binding.

Distinct subfamilies of primate L1Gg retroposons, with some elements carrying tandem repeats in the 5' region.

Two subfamilies of L1 elements, differing dramatically in the first 1.2 kb of sequence at their 5' ends, were identified in the prosimian primate, Galago garnetti. Interesting patterns of sequence similarity were observed between the galago subfamilies, and with the L1s from human and from another prosimian, the slow loris. Furthermore, members of one of the subfamilies have six to eight tandemly repeated units of 73 bp, starting about 730 bp from their 5' ends. Such tandem repeats have not been reported in other primate L1s, but a striking sequence similarity was found between the galago tandem repeats and those previously described at the 5' termini of some mouse L1s [Loeb, D. D. et al. Mol. Cell. Biol. 6, 168-182, 1986]. Although the similar sequence indicates a shared, conserved function, the galago repeats are sub-terminal and therefore cannot serve as portable RNA polymerase II promoters, as has been suggested for the mouse tandem repeats.