Beta-chain Gene Research Articles

Modification of the Interleukin-6 Response to Air Pollution by Interleukin-6 and Fibrinogen Polymorphisms

BackgroundEvidence suggests that cardiovascular effects of air pollution are mediated by inflammation and that air pollution can induce genetic expression of the interleukin-6 gene (IL6).ObjectivesWe investigated whether IL6 and fibrinogen gene variants can affect plasma IL-6 responses to air pollution in patients with cardiovascular disease.MethodsWe repeatedly determined plasma IL-6 in 955 myocardial infarction survivors from six European cities (n = 5,539). We conducted city-specific analyses using additive mixed models adjusting for patient characteristics, time trend, and weather to assess the impact of air pollutants on plasma IL-6. We pooled city-specific estimates using meta-analysis methodology. We selected three IL6 single-nucleotide polymorphisms (SNPs) and one SNP each from the fibrinogen α-chain gene (FGA) and β-chain gene (FGB) for gene–environment analyses.ResultsWe found the most consistent modifications for variants in IL6 rs2069832 and FBG rs1800790 after exposure to carbon monoxide (CO; 24-hr average; p-values for interaction, 0.034 and 0.019, respectively). Nitrogen dioxide effects were consistently modified, but p-values for interaction were larger (0.09 and 0.19, respectively). The strongest effects were seen 6–11 hr after exposure, when, for example, the overall effect of a 2.2% increase in IL-6 per 0.64 mg/m3 CO was modified to a 10% (95% confidence interval, 4.6–16%) increase in IL-6 (p-value for interaction = 0.002) for minor homozygotes of FGB rs1800790.ConclusionsThe effect of gaseous traffic-related air pollution on inflammation may be stronger in genetic subpopulations with ischemic heart disease. This information could offer an opportunity to identify postinfarction patients who would benefit more than others from a cleaner environment and antiinflammatory treatment.

Primary Cutaneous Peripheral T-Cell Lymphoma With Aberrant Coexpression of CD20: Case Report and Review of the Literature

We report an unusual case of primary cutaneous T-cell lymphoma in a 45-year-old male showing expression of the B-cell marker CD20. The lesion presented as a large plaque on the right shin and rapidly developed into additional lesions on the right lower leg and right anterior chest. Despite therapy, bone involvement was discovered at 1 year after initial presentation. Punch biopsy of the right leg revealed a diffuse, atypical dermal lymphocytic infiltrate with papillary edema and no epidermotropism. The atypical cells strongly expressed the T-cell markers CD3, CD2, and CD43, with additional expression of cytotoxic T-cell markers CD8, cytotoxic granule-associated RNA binding protein (TIA-1), and granzyme B and loss of CD7 by immunohistochemistry (IHC). In addition, the atypical cells with the above phenotype exhibited moderate expression of CD20 by IHC. Coexpression of CD8 and CD20 in the atypical cells was confirmed by a dual-labeled IHC stain. Clonal rearrangements of the T-cell receptor gamma- and beta-chain genes were detected by polymerase chain reaction; however, there was no expression of T-cell receptor beta-chain identified by IHC. In conclusion, we present a case a of CD8-positive primary cutaneous T-cell lymphoma with CD20 expression, exhibiting aggressive behavior.

CD20-Positive T-Cell Large Granular Lymphocyte Leukemia: Case Report and Review of the Literature

CD20-positive T-cell malignancy is a rare disease. We report a case of CD20-positive T-cell large granular lymphocyte leukemia (T-LGLL). The leukemic cells were positive for CD20 and T cell markers, such as CD3, CD4, CD5, CD8 and CD57. A monoclonal rearrangement of the T-cell receptor (TCR) beta chain gene was detected. Twenty-three cases of well-documented CD20-positive T-cell malignancies were reviewed. Most cases were mature T-cell malignancies, especially exhibiting a cytotoxic T-cell phenotype, despite a diversity of the pathological diagnoses. Additional cases must be evaluated to clarify the implications of CD20 expression on T-cell malignancies and to elucidate whether such cases constitute a distinct biologic and clinical disease entity. The accumulation of cases will help to facilitate provision of a proper treatment for CD20-positive T-cell malignancies in the future.

Activation of Dormant Bacterial Genes byNonomuraeasp. Strain ATCC 39727 Mutant-Type RNA Polymerase

There is accumulating evidence that the ability of actinomycetes to produce antibiotics and other bioactive secondary metabolites has been underestimated due to the presence of cryptic gene clusters. The activation of dormant genes is therefore one of the most important areas of experimental research for the discovery of drugs in these organisms. The recent observation that several actinomycetes possess two RNA polymerase beta-chain genes (rpoB) has opened up the possibility, explored in this study, of developing a new strategy to activate dormant gene expression in bacteria. Two rpoB paralogs, rpoB(S) and rpoB(R), provide Nonomuraea sp. strain ATCC 39727 with two functionally distinct and developmentally regulated RNA polymerases. The product of rpoB(R), the expression of which increases after transition to stationary phase, is characterized by five amino acid substitutions located within or close to the so-called rifampin resistance clusters that play a key role in fundamental activities of RNA polymerase. Here, we report that rpoB(R) markedly activated antibiotic biosynthesis in the wild-type Streptomyces lividans strain 1326 and also in strain KO-421, a relaxed (rel) mutant unable to produce ppGpp. Site-directed mutagenesis demonstrated that the rpoB(R)-specific missense H426N mutation was essential for the activation of secondary metabolism. Our observations also indicated that mutant-type or duplicated, rpoB often exists in nature among rare actinomycetes and will thus provide a basis for further basic and applied research.

Influence of fibrinogen β-chain gene variations on risk of myocardial infarction in a Chinese Han population

Although the role of fibrinogen as a predictor of acute myocardial infarction (MI) has been well-established, the association of genetic polymorphisms in the fibrinogen gene with MI is still controversial. This study was conducted to elucidate the association between the genetic polymorphisms of the fibrinogen beta-chain (FGB) gene and MI in Chinese Han population. The occurrence of 3 common polymorphisms (i.e., -455G/A, R448K and 8558C/G) in a case-control study including 508 patients with MI and 503 healthy controls was investigated. Results Analyses of single polymorphisms showed that individuals carrying the rare alleles for the 3 polymorphisms were significantly associated with a decreased risk of MI. Logistic regression analysis indicated that R448K remained independently associated with MI after adjustment for environmental risk factors (adjusted odds ratio(OR) = 0.71 for KK/RK versus RR, P = 0.023). The three polymorphisms were found to be in strong linkage disequilibrium. Haplotype analyses showed that the A-K-G haplotype (-455A, 448K, 8558G) was associated with a protective effect against MI. Compared with the common haplotype G-R-C, the adjusted OR for A-K-G was 0.68 (95% CI, 0.51-0.90; P = 0.006). These data indicate that individuals carrying the FGB 448K allele may be protective against having MI in this population.

Susceptibility to type I diabetes in women is associated with the CD3 epsilon locus on chromosome 11

Type I diabetes is associated with the DQ loci of the MHC and to a lesser extent with the T cell antigen receptor (TcR) beta chain genes. The non-obese diabetic (NOD) mouse is an animal model of human diabetes, in which up to 90% of female mice develop overt insulin-dependent diabetes. Genetic studies in the NOD mouse suggest that there are at least three diabetogenic genes; one that maps to the MHC, another that may map to the mouse Thy-I locus, and a third that has still to be identified. We have investigated loci in the vicinity of the human Thy-I locus on chromosome 11q23 and report here the results of restriction fragment length polymorphism (RFLP) analysis of the CD3 epsilon locus of 168 Caucasoid patients with type I diabetes. While no association was found between this locus and type I diabetes, a significant difference in the frequency of the CD3 epsilon 8-kb allele was found between male and female patients (0.268 versus 0.430; P less than 0.0025, Pc = 0.02) and between female patients and healthy female controls (0.430 versus 0.267; P less than 0.015). These results suggest that a gene residing on chromosome 11q23 may confer susceptibility to type I diabetes in women.

HLA-DR, DQ and T cell antigen receptor constant beta genes in Japanese patients with ulcerative colitis

We studied the T cell antigen receptor (TcR) constant beta chain genes on HLA typed Japanese patients with ulcerative colitis (UC). A TcR constant beta EcoRI 6.0-kb fragment was present in all Japanese UC patients (n = 17) but completely absent in the controls (n = 35) (chi2 = 47.6, P less than 0.001). The frequency of HLA-DR2 antigen was significantly higher in UC patients (85% versus 28% in controls, P less than 0.001). Furthermore, HLA-DQw1 antigen was also increased in UC patients (96% versus 60% in controls, P less than 0.001). However, HLA-DR4 antigen was significantly decreased in UC patients (12% versus 37%, P = 0.02). HLA-DR1 antigen was not found in UC patients and was present in only 15% of the controls. These results suggest that TcR beta chain and HLA-DQw1 antigen may be important in the pathogenesis of Japanese UC.

Lack of association of T cell receptor beta-chain constant region polymorphism with insulin-dependent diabetes mellitus in Finland

Allelic polymorphism in the T cell receptor constant beta-chain gene region has been reported to be associated with autoimmune diseases, including insulin-dependent diabetes mellitus (IDDM). The present analysis of 164 children and adolescents with IDDM and 193 controls for BqlII polymorphism using a TcR-C beta cDNA probe revealed two allelic restriction fragments with sizes of 10.5 kb (U) and 9.6 kb (L). No particular association was observed between the RFLP genotypes and IDDM (UU 27% versus 31%; UL 53% versus 52%; and LL 20% versus 17%, in diabetic subjects and controls, respectively), nor were any differences found between patients with various HLA risk antigens. The frequency of heterozygotes was 52% in 63 DR3-positive diabetic subjects and 53% in 73 DR3-negative ones. The results do not support any involvement of the TcR constant region genes in susceptibility to IDDM.

Enhanced functionality of T cell receptor-redirected T cells is defined by the transgene cassette

The transfer of T cell receptor (TCR) genes allows to endow T cells with a new antigen specificity. For clinical applications of TCR-redirected T cells, efficient functional expression of the transgenic TCR is a key prerequisite. Here, we compared the influence of the transgene cassette on the expression and function of the murine TCR P14 (recognizing a LCMV gp33 epitope) and the human TCR WT-1 (recognizing an epitope of the tumor-associated antigen WT-1). We constructed different vectors, in which TCRalpha- and beta-chain genes were either (a) linked by an internal ribosomal entry site (IRES), (b) combined by a 2A peptide, or (c) introduced into two individual retroviral constructs. While in a TCR-deficient T cell line TCR P14 was expressed equally well by all constructs, we found that IRES- but not 2A-employing TCR expression is hampered in a TCR-bearing cell line and in primary murine T cells where the transgenic TCR has to compete with endogenous TCR chains. Similarly, 2A-linked TCR WT-1 genes yielded highest expression and function as measured by tetramer binding and peptide-specific IFN-gamma secretion. Differences in expression were independent of copy number integration as shown by real-time PCR. Thus, linking TCRalpha- and beta-chain genes by a 2A peptide is superior to an IRES for TCR expression and T cell function.

Role of human non-invariant NKT lymphocytes in the maintenance of type 2 T helper environment during pregnancy

The molecular and cellular mechanisms that generate the T(h)2 cytokine environment necessary for the maintenance of pregnancy are still not fully understood. We herein show that the human decidua is highly enriched for TCR alpha beta(+)CD161(+) NKT cells. They express non-invariant antigen receptors encoded by diverse TCRV alpha- and V beta-chain gene segments, thereby referred to as non-invariant NKT (non-iNKT) cells. In spite of their diverse TCR expression, they do not recognize fetal allo-antigens but specifically responded to CD1d-transfected cell lines. In contrast to the peripheral blood non-iNKT cells, the decidua-residing non-iNKT cells had a marked T(h)2 bias. In addition, they suppress the mixed leukocyte reaction directed against the paternal antigens. The T(h)2 cytokines have been known to stimulate trophoblast outgrowth and invasion. Thereby, the non-iNKT cells residing in the decidual tissue may have a functionally important interaction with the villous and extravillous trophoblast cells expressing CD1d and may therefore play a pivotal role in successful pregnancy by inhibiting fetal rejection and enhancing placental growth. These findings may reflect one mechanism that is an essential component for the T(h)2 environment necessary for the maintenance of pregnancy.

A Polymorphism of Fibrinogen Beta Chain (FGB) Gene is Not Associated with Autistic Spectrum Disorder in Korean Population

Evidences has been accumulated the difference of cardiovascular phenotypes in autistic spectrum disorder (ASD). To determine the genetic association between fibrinogen beta chain (FGB) gene and ASD in Korean population, we genotyped single nucleotide polymorphism (SNP) (rs4220, Arg478Lys, exon 8) in the FGB gene by using direct sequencing. Among nonsynonymous SNPs in the coding region of FGB, only one SNP's heterozygosity (rs4220) is more than 0.05. Therefore, we analyzed the association between rs4220 and ASD. Three hundred six control and 196 ASD subjects were evaluated. For the analysis of genetic data, SNPStats, SNPAnalyzer, and Helixtree programs were used. Multiple logistic regression analysis (codominant, dominant, and recessive models) was also used. The result showed that a SNP (rs4220) in the FGB gene was not significantly difference between ASD and controls in three alternative models. This result suggests that the FGB gene may have no relation to the development of ASD.

LMP2-Specific TCR Gene Therapy for Hodgkin Lymphoma - Design of a Dominant TCR Construct for Immunotherapy.

Hodgkin lymphoma (HL) is the second most commonly diagnosed cancer in the 15–29 year old population. EBV-positive Hodgkin lymphoma typically demonstrates latency II antigen expression, characterised by loss of most EBV antigens except for the latent membrane protein (LMP) 1 and 2 and the EBNA-1 protein. LMP2 is expressed in Reed Sternberg cells and may serve as a target for antigen-specific immunotherapy. However, LMP2 is poorly immunogenic and it is often difficult to generate autologous LMP2-specific cytotoxic T lymphocytes (CTL) for adoptive immunotherapy. T cell receptor (TCR) gene transfer using retroviral vectors containing the TCR alpha and beta chain genes can reproducibly redirect the antigen specificity of a given population of T cells. Such an approach has been used here to generate LMP2-specific CTL independent of the immuno-competence of the patient. The goal of this study was to generate a retroviral TCR construct suitable for rapid and efficient production of LMP2-specific CTL. Retrovirally introduced TCRs compete with endogenous TCRs for a limited pool of CD3 molecules required for assembly of the TCR complex. Competition for CD3 molecules may limit surface expression of the introduced TCR resulting in a transduced T cell with poor functional avidity. In an attempt to generate a ‘highly competitive' LMP2-TCR the following modifications were made to the retroviral vector construct:i.nucleotide sequences were codon optimised for efficient translation in human cells;ii.the TCR chain constant regions were altered to contain murine sequences to enhance CD3 binding; andiii.the TCR alpha and beta chain genes were linked by a self-cleaving 2A sequence from the porcine teschovirus to enhance equimolar expression of both TCR chains.The unmodified HLA-A2-restricted LMP2-specific TCR was poorly expressed in primary human T cells, suggesting that it competed inefficiently with endogenous TCR chains for cell surface expression. Very few CD8+Vβ13+ T cells were detectable after LMP2-TCR transduction (up to 2.5% of viable CD3+ T cells, as detected by FACs analysis using monoclonal anti-Vβ13 antibodies), which included 1.9% CD8+ T cells expressing endogenous Vβ13+ TCRs as quantified in mock-transduced control cells. Poor expression of the wild type LMP2-TCR was consistently observed in independent transduction experiments. However, transduction with the modified LMP2-TCR construct resulted in cell surface expression of the TCR in 55–65% viable CD3+ T cells. HLA-A2/LMP2 pentamer binding was demonstrated in 36–39% CD8+ CTL cells immediately post transduction. The transduced cells showed peptide-specific IFNγ and IL2 production and killed target cells displaying the LMP2 peptide. Of major importance, expression of the introduced LMP2-TCR completely suppressed the cell surface expression of almost the entire repertoire of endogenous TCR combinations, including ‘mis-paired' TCRs in the transduced primary human T cells. ‘Mis-paired' TCRs contain an introduced alpha chain paired with an endogenous beta chain and vice versa. The antigen specificity of such mispaired TCRs generated after transduction is unknown and could lead to unwanted side effects. The design of vectors containing modified TCR sequences, which produce ‘dominant' TCRs may improve the efficacy of TCR gene therapy and reduce the risk of potential auto-reactivity of endogenous and ‘mis-paired' TCR combinations. We have shown that LMP2-specific T cells can be readily generated by TCR gene transfer with minimal risk of autoreactivity.

Monoclonal anti-Ia antibody therapy in animal models of autoimmune disease.

Allele-specific monoclonal anti-I-A antibodies are capable of specifically suppressing the immune response to antigens under the control of the allele towards which the antibody is directed, without suppressing the response to antigens under the control of the alternative allele of the I-A alpha and beta chain genes in an F1 heterozygote. This phenomenon, which has been termed 'allele-specific immunosuppression', is antigen-specific, long-lasting and transferrable with Thy-1-positive spleen cells. This type of immunosuppression has been applied to animal models of autoimmune disease, in both homozygous and heterozygous animal models. Anti-I-A monoclonal antibodies are capable of preventing, suppressing and treating experimental allergic encephalomyelitis (EAE), of partially suppressing experimental autoimmune myasthenia gravis, and of preventing the onset of type I insulin-dependent diabetes in the BB/W diabetic rat. In addition, this type of immunotherapy has succeeded in almost completely suppressing nephritis in NZB X NZW F1 mice, which normally develop severe lupus-like nephritis. Significant toxicity, which may be due to anti-allotype antibodies, anti-idiotype antibodies, or to impurities in the monoclonal antibody preparations, has been encountered in the BB/W diabetic rat. In addition, attempts to extend these observations to EAE in the cynomolgus monkey have encountered significant mortality which appears to be attributable to the monoclonal antibody injections (anti-HLA-DR). The mechanism of this toxicity and means of circumventing it are currently under investigation. These results demonstrate the critical role of I-A molecules in the induction and continuance of the autoimmune process in these experimental animal models.

Detection of COL1A1-PDGFB fusion transcripts and PDGFB/PDGFRB mRNA expression in dermatofibrosarcoma protuberans

Fusion of the collagen type I alpha 1 (COL1A1) gene with the platelet-derived growth factor beta chain (PDGFB) gene has been described in dermatofibrosarcoma protuberans. The abnormal fusion transcripts probably cause PDGFB and its receptor (platelet-derived growth factor receptor beta, PDGFRB) autocrine stimulation and cell proliferation, which are responsible for the development of dermatofibrosarcoma protuberans. A reverse transcription–polymerase chain reaction assay was performed to detect the COL1A1-PDGFB fusion transcripts in 57 samples. In addition, the PDGFB gene amplification and PDGFB/PDGFRB mRNA levels were quantified by a real-time PCR system for the samples in which the fusion transcripts had been successfully detected. The fusion transcripts were detected in 42 of 57 samples. Various exons of the COL1A1 gene were fused in frame with the PDGFB gene; exons 7 and 25 were found to be slightly more frequently involved than the other exons. The PDGFB gene amplification levels varied from 0.6 to 8.3 (mean 2.4) in 42 tumor samples and from 0.4 to 3.0 (mean 1.2) in 20 adjacent normal tissue samples. In the 20 paired samples, the PDGFB gene amplification in the tumor was significantly higher than that in the normal tissue. The presence of PDGFB and PDGFRB mRNAs was demonstrated in 26 and 21 of 26 cases, respectively. The PDGFB and PDGFRB mRNA expression levels showed a good correlation (r=0.76, P<0.0001). These results indicate that the fusion protein, which is processed by the COL1A1-PDGFB transcripts, can serve as a functional ligand for PDGFRB.

Cloning and large-scale expansion of epitope-specific equine cytotoxic T lymphocytes using an anti-equine CD3 monoclonal antibody and human recombinant IL-2

Cytotoxic T lymphocytes are involved in controlling intracellular pathogens in many species, including horses. Particularly, CTL are critical for the control of equine infectious anemia virus (EIAV), a lentivirus that infects horses world-wide. In humans and animal models, CTL clones are valuable for evaluating the fine specificity of epitope recognition, and for adoptive immunotherapy against infectious and neoplastic diseases. Cloned CTL would be equally useful for similar studies in the horse. Here we present the first analysis of a method to generate equine CTL clones. Peripheral blood mononuclear cells were obtained from an EIAV-infected horse and stimulated with the EIAV Rev-QW11 peptide. Sorted CD8+ T cells were cloned by limiting dilution, and expanded without further antigen addition using irradiated PBMC, anti-equine CD3, and human recombinant IL-2. Clones could be frozen and thawed without detrimental effects, and could be subsequently expanded to numbers exceeding 2 × 10 9 cells. Flow cytometry of expanded clones confirmed the CD3+/CD8+ phenotype, and chromium release assays confirmed CTL activity. Finally, sequencing TCR beta chain genes confirmed clonality. Our results provide a reliable means to generate large numbers of epitope-specific equine CTL clones that are suitable for use in downstream applications, including functional assays and adoptive transfer studies.

Pulmonary Hypertension in a Case of Hb-Mainz Hemolytic Anemia

The development of pulmonary arterial hypertension (PAH) is the leading cause of mortality in patients with thalassemia and sickle cell anemia and was reported to occur in hemolytic anemias such as hereditary stomatocytosis, and paroxysmal nocturnal hemoglobinuria. Here, we report for the first time on the development of PAH in a patient with Hb-Mainz hemolytic anemia. Hb-Mainz is an unstable hemoglobin variant resulting from mutations at codon 98 of the beta chain gene (Val>Glu) characterized by severe chronic hemolytic anemia. The development of PAH in this patient further supports the contention that there is a clinical syndrome of hemolysis-associated development of PAH.

Análisis del gen de fusión COL1A1-PDGFB en un caso de dermatofibrosarcoma protuberans con componente de fibrosarcoma

Dermatofibrosarcoma protuberans (DFSP) is an infrequent tumor of intermediate malignancy, with little tendency to develop metastases but with a high rate of local recurrence. Cytogenetically, DFSP is characterized by a reciprocal translocation, t(17;22)(q22;q13), which is a conditioning factor in the fusion of the collagen type I alpha I gene (COL1A1) in chromosome 17q with the platelet-derived growth factor beta chain gene (PDGFB) in chromosome 22q. The fusion of these genes is variable, involving one of the 51 exons of the COL1A1 gene and exon 2 of the PDGFB gene. We present the case of a 37-year-old woman with a tumor on the arm whose histology showed a neoplastic infiltration of the subcutaneous cellular tissuemade up of fusiform cells with an elongated nucleus in a storiform pattern and other more pleomorphic cells in a herringbone pattern, compatible with DFSP with a fibrosarcoma component. The molecular biology study with RT-PCR analysis of paraffin-embedded material and later sequencing showed a new fusion of exon 19 of the COL1A1 gene and exon 2 of PDGFB, supporting a diagnosis of DFSP. A study of the COL1A1-PDGFB fusion products is useful in cases where histology and immunohistochemistry are insufficient for the differential diagnosis of DFSP versus other sarcomas. It also justifies the use of new avenues of treatment with tyrosine kinase inhibitors.

Clonally Expanded T-Cells in the Peripheral Blood of Patients with Idiopathic Thrombocytopenic Purpura and Helicobacter pylori Infection

Eradication of Helicobacter pylori leads to platelet recovery in some patients with idiopathic thrombocytopenic purpura (ITP). Therefore, the pathogenesis of a subgroup of ITP is probably associated with H pylori infection (H pylori-related ITP). If H pylori-related ITP is a definite subgroup of ITP, specific oligoclonal T-cells might accumulate in the peripheral blood (PB). To address this issue, we performed single-strand conformation polymorphism analysis of complementarity-determining region 3 (CDR3) of the T-cell receptor beta-chain genes of PB T-cells. Fourteen ITP patients with H pylori infection and 12 ageadjusted healthy volunteers were studied. Of the 14 patients, 8 patients (responders) exhibited a platelet response after successful H pylori eradication therapy, but 6 patients (nonresponders) did not. Vbeta5.2, Vbeta15, and Vbeta19 gene usage by clonally expanded T-cells in PB obtained before H pylori eradication therapy was significantly higher in responders than in nonresponders or healthy volunteers (Vbeta5.2, P = .023; Vbeta15, P = .004; Vbeta19, P = .036). Furthermore, an abrogation of clonally expanded T-cells was observed after therapy in some responders. These findings suggest that specific T-cell clones accumulate in H pylori-related ITP and that such clones may be associated with immune-mediated destruction of platelets.

Reversion of the biochemical defects in murine embryonic Sandhoff neurons using a bicistronic lentiviral vector encoding hexosaminidase α and β

Sandhoff disease, a neurodegenerative disorder characterized by the intracellular accumulation of GM2 ganglioside, is caused by mutations in the hexosaminidase beta-chain gene resulting in a hexosaminidase A (alphabeta) and B (betabeta) deficiency. A bicistronic lentiviral vector encoding both the hexosaminidase alpha and beta chains (SIV.ASB) has previously been shown to correct the beta-hexosaminidase deficiency and to reduce GM2 levels both in transduced and cross-corrected human Sandhoff fibroblasts. Recent advances in determining the neuropathophysiological mechanisms in Sandhoff disease have shown a mechanistic link between GM2 accumulation, neuronal cell death, reduction of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) activity, and axonal outgrowth. To examine the ability of the SIV.ASB vector to reverse these pathophysiological events, hippocampal neurons from embryonic Sandhoff mice were transduced with the lentivector. Normal axonal growth rates were restored, as was the rate of Ca(2+) uptake via the SERCA and the sensitivity of the neurons to thapsigargin-induced cell death, concomitant with a decrease in GM2 and GA2 levels. Thus, we have demonstrated that the bicistronic vector can reverse the biochemical defects and down-stream consequences in Sandhoff neurons, reinforcing its potential for Sandhoff disease in vivo gene therapy.

Analysis of T-Cell Receptor Gene Rearrangement for Predicting Clinical Outcome in Patients With Cutaneous T-Cell Lymphoma

To extend previous observations regarding the prognostic value of analyzing lymph node DNA from patients with cutaneous T-cell lymphoma for the presence of a monoclonal T-cell population by Southern blot vs polymerase chain reaction (PCR) methods. Inception cohort study from 1982 to 1998. Recruitment of new patients ended in 1994. A tertiary care referral center in Seattle, Wash. Patients Fifty-five uniformly staged patients with the diagnosis of cutaneous T-cell lymphoma who underwent a lymph node biopsy, 21 with clinically abnormal nodes and 34 with normal nodes. Interventions Lymph nodes were evaluated for T-cell receptor (TCR) gamma-chain gene rearrangement by 2 PCR methods: capillary electrophoresis and denaturing gradient gel electrophoresis. The same lymph nodes were evaluated by Southern blot analysis for TCR beta-chain gene rearrangement and examined histopathologically on the basis of the National Cancer Institute lymph node classification system. Patients were observed clinically for a mean of 9.5 years. Skin stage, clinical lymph node examination, lymph node histologic examination, Southern blot analysis, and PCR analyses were evaluated as potential prognostic predictors by univariate and multivariate analyses. The statistical association of TCR analysis and clinical outcome was determined among all patients. Hazard ratios (HRs) by Cox proportional hazards regression analysis were used to estimate the risk of a poor clinical outcome. Cumulative survival rates were analyzed by the Kaplan-Meier method. A skin stage of T3 (tumors) or T4 (erythroderma) was the most powerful predictor of a poor clinical outcome (HR, 31.3 vs T1; P<.001). Patients with detectable TCR gamma-chain gene rearrangement in lymph node DNA by PCR also were more likely to have a poor outcome (HR, 5.1; P<.001), but it was a less powerful predictor than skin stage. Even when the skin stage, presence or absence of lymphadenopathy, and histologic lymph node score were known for the patient, Southern blot analysis still added to prediction of a poor outcome (HR, 9.3; P = .007), whereas PCR provided no statistically significant additional information on outcome. Detection of a monoclonal T-cell population by PCR in lymph nodes of patients with cutaneous T-cell lymphoma does not enhance prediction of clinical outcome and probability of survival beyond what can be determined from clinical examination and histologic lymph node scores. Skin stage and the presence or absence of lymphadenopathy remain the most important determinants of clinical outcome.