Selective correlations; not voodoo.

The problem of "voodoo" correlations-exceptionally high observed correlations in selected regions of the brain-is well recognized in neuroimaging. It arises when quantities of interest are estimated from the same data that was used to select them as interesting. In statistical terminology, the problem of inference following selection from the same data is that of selective inference. Motivated by the unwelcome side-effects of splitting the data- the recommended remedy-we adapt the recent developments in selective inference in order to construct confidence intervals (CIs) with good reproducibility prospects, even if selection and estimation are done with the same data. These intervals control the expected proportion of non-covered correlations in the selected voxels-the False Coverage Rate (FCR). They extend further toward zero than standard intervals, thus attenuating the impression made by highly biased observed correlations. They do so adaptively, in that they coincide with the standard CIs when far away from the selection point. We complement existing analytic proofs with a simulation, showing that the proposed intervals control the FCR in realistic social neuroscience problems. We also suggest a "confidence calibration plot", to allow the intervals to be reported in a clear and interpretable way. Applying the proposed methodology on a loss-aversion study, we demonstrate that with the sample size and selection type employed, selection bias is considerable. Finally, selective intervals are compared to the currently recommended data-splitting approach. We discover that our approach has more power and typically more informative, as no data is discarded. Computation of the intervals is implemented in an accompanying software package.

Revisiting multi-subject random effects in fMRI: advocating prevalence estimation.

Random effect analysis has been introduced into fMRI research in order to generalize findings from the study group to the whole population. Generalizing findings is obviously harder than detecting activation within the study group since in order to be significant, an activation has to be larger than the inter-subject variability. Indeed, detected regions are smaller when using random effect analysis versus fixed effects. The statistical assumptions behind the classic random effect model are that the effect in each location is normally distributed over subjects, and "activation" refers to a non-null mean effect. We argue that this model is unrealistic compared to the true population variability, where due to function-anatomy inconsistencies and registration anomalies, some of the subjects are active and some are not at each brain location. We propose a Gaussian-mixture-random-effect that amortizes between-subject spatial disagreement and quantifies it using the prevalence of activation at each location. We present a formal definition and an estimation procedure of this prevalence. The end result of the proposed analysis is a map of the prevalence at locations with significant activation, highlighting activation regions that are common over many brains. Prevalence estimation has several desirable properties: (a) It is more informative than the typical active/inactive paradigm. (b) In contrast to the usual display of p-values in activated regions - which trivially converge to 0 for large sample sizes - prevalence estimates converge to the true prevalence.

The x gene is essential for HTLV replication.

The human T-cell leukemia viruses (HTLV) are associated with T-cell malignancies in man and will transform normal human T cells in vitro. The mechanism of malignant transformation by HTLV is unknown but appears to be distinct from that of other classes of retroviruses, which induce malignant transformation through viral or cellular oncogenes. Recently a new gene, termed x, was identified in HTLV. This gene has been hypothesized to be the transforming gene of HTLV because of its conservation within the HTLV class of retroviruses. By in vitro mutagenesis of the HTLV-II x gene, it is now demonstrated that the presence of a functional x gene product is necessary for efficient HTLV transcription. Therefore, these studies provide direct evidence for an important function of the x gene in HTLV replication. The functional analogies between the x gene and transcriptional regulatory genes of some DNA viruses suggest that these viruses share similar mechanisms for cellular transformation.

High rate of HTLV-II infection in seropositive i.v. drug abusers in New Orleans.

Confirmed infection with HTLV-II (human T cell leukemia virus type II) has been described only in rare cases. The major limitation to serological diagnosis of HTLV-II has been the difficulty of distinguishing HTLV-II from HTLV-I (human T cell leukemia virus type I) infection, because of substantial cross-reactivity between the viruses. A sensitive modification of the polymerase chain reaction method was used to provide unambiguous molecular evidence that a significant proportion of intravenous drug abusers are infected with HTLV, and the majority of these individuals are infected with HTLV-II rather than HTLV-I. Of 23 individuals confirmed by polymerase chain reaction analysis to be infected with HTLV, 21 were identified to be infected with HTLV-II, and 2 were infected with HTLV-I. Molecular identification of an HTLV-II--infected population provides an opportunity to investigate the pathogenicity of HTLV-II in humans.

HTLV-II transactivation is regulated by the overlapping tax/rex nonstructural genes.

The human T-cell leukemia virus (HTLV) types I and II have two nonstructural genes that are encoded in overlapping reading frames. One of these genes, known as tax, has been shown to encode a protein responsible for enhanced transcription (transactivation) from the viral long terminal repeats (LTRs). Genetic evidence indicates that the second nonstructural gene of HTLV-II, here designated rex, acts in trans to modulate tax gene-mediated transactivation in a concentration-dependent fashion. The rex gene may regulate the process of transactivation during the viral life cycle.

HTLV-I and HTLV-II tax trans-activate the human EGR-1 promoter through different cis-acting sequences.

The immediate-early response gene, EGR-1, encodes a zinc finger-containing transcription factor that is involved in growth and differentiation of a variety of cell types. EGR-1 is induced in normal T cells following mitogenic stimulation and has recently been shown to be constitutively expressed in human T-cell leukemia virus type I (HTLV-I)- and type II (HTLV-II)-transformed T-cell lines. The trans-activating protein of HTLV-I, Tax, has been demonstrated to trans-activate promoters of a number of cellular genes, some of which may be critical in regulating T-cell proliferation. In this study, we examine the effect of Tax on expression of EGR-1 in three T-cell lines and demonstrate that both HTLV-I and -II Tax are capable of trans-activating human EGR-1 recombinant promoter constructs. Interestingly, HTLV-I and -II Tax trans-activate the human EGR-1 promoter through different promoter regions in the Jurkat cell line, suggesting that HTLV-I and -II Tax may lead to constitutive expression of EGR-1 through different signaling pathways. Deregulated expression of EGR-1 may contribute to uncontrolled cell growth and transformation during early stages of T-cell activation in HTLV-I and -II-infected cells.

Cell type-specific activity of the N-myc promoter in human neuroblastoma cells is mediated by a downstream silencer.

The N-myc oncogene is actively transcribed in many neuroblastoma tumors, but is not expressed in mature, normal tissue of any type. Chloramphenicol acetyl transferase (CAT) assays of constructs containing N-myc sequence transfected into N-myc expressing LA-N-5 neuroblastoma cells or non-expressing HeLa carcinoma cells have revealed a 201 base pair (bp) regulatory region mediating the cell type-specific activity of the promoter. While located downstream from 5' mRNA cap sites, the region appears to function by preventing transcriptional initiation. This downstream region is capable of suppressing promoter activity independently of position, and contains an element having 100% homology with the 9 bp consensus sequence of a transcriptional silencer found in the upstream region of the lysozyme gene. DNA gel retardation assays have shown that this sequence is involved in a specific DNA-protein interaction with nuclear extract from HeLa cells that is distinct from that occurring with extract from LA-N-5 cells. These results suggest that the N-myc promoter's cell type-specific activity is regulated by a downstream silencer, and that differential binding of regulatory protein from that present in non-expressing cells may result in the constitutive N-myc expression seen in neuroblastoma.

Activation of the GM-CSF promoter by HTLV-I and -II tax proteins.

Production of granulocyte-macrophage colony-stimulating factor (GM-CSF) by normal T lymphocytes requires activation by antigen, mitogen or lectin, whereas T-cell lines transformed by human T-cell leukemia virus type I (HTLV-I) or type II (HTLV-II) constitutively produce high levels of GM-CSF. Using transient cotransfection assays, we demonstrate that introduction of the tax gene of either HTLV-I or HTLV-II is sufficient to activate GM-CSF promoter constructs in an unstimulated T-cell line. The GM-CSF 5' flanking sequences previously shown to be sufficient for GM-CSF induction following T-cell activation are also sufficient for activation by the HTLV tax proteins. The sequences required for trans-activation of GM-CSF are distinct from those required for the activation of other T-cell-inducible genes (IL-2R alpha, IL-2) by tax, suggesting that tax can have pleiotropic effects on gene expression in T cells. Constitutive GM-CSF production by HTLV-infected T cells may therefore be due to trans-activation of its promoter by tax. Expression of GM-CSF by HTLV-I infected lymphocytes may be important in the granulocytosis and eosinophilia frequently seen in patients with HTLV-I-induced adult T-cell leukemia/lymphoma.

Cell type-specific expression and negative regulation by retinoic acid of the human N-myc promoter in neuroblastoma cells.

Expression of the N-myc oncogene is an important determinant of tumor behavior in human neuroblastoma. To study the regulation of N-myc, we have subcloned fragments of the 5' flanking region of the human N-myc gene upstream of the chloramphenicol acetyl transferase (CAT) reporter gene, and assayed for promoter activity in transient transfections into neuroblastoma and other cell lines. Upstream sequences were found to possess promoter activity to within 121 bp of the major cap site (-121). Negative regulatory elements were identified in regions approximately 2 kb and 500 bp upstream from the major cap site, as well as 150-1000 bp downstream. Promoter constructs containing downstream elements from bp +150 to +1000 were active in N-myc-expressing neuroblastoma cell lines, but not in non-expressing Epstein-Barr virus (EBV)-transformed 729-6 B-cell or HeLa cell lines, while those lacking this element were active in all cell types tested. All tested constructs retaining promoter activity showed decreased activity in parallel with the down-regulation of endogenous N-myc in response to treatment of transfected cells with retinoic acid. These studies suggest that N-myc regulation may be controlled at different levels, and provide a basis for further characterization of N-myc regulation in neuroblastoma.

Transactivation of cellular genes by human retroviruses.

We have focused this chapter on interactions with two of the best characterized transregulatory genes, tax for HTLV-I/II and Tat for HIV-1. Both genes illustrate the complex interplay between retroviral regulatory genes and cellular gene regulation. In both instances a viral gene of relatively straightforward function in the viral context appears to cause extensive dysregulation of cellular genes, either directly or as a consequence of altered cellular differentiation. Understanding this viral/cellular gene cross-talk may elucidate mechanisms leading to malignant transformation autoimmune disease and to neurologic and paraneoplastic complications such as hypercalcemia for HTLV-I/II, as well as the pathogenesis of immune dysfunction and opportunistic malignancy in HIV-I/II-infected individuals. An understanding of functional mechanisms of these transregulatory viral genes will undoubtedly afford better explanations for the myriad manifestations of retroviral infection.

Effects of the farnesyl transferase inhibitor R115777 on normal and leukemic hematopoiesis.

Patients with acute myelogenous leukemia or myelodysplastic syndrome may respond to farnesyl transferase inhibitors (FTIs) with partial or complete response rates noted in about 30% of such patients. FTIs prevent the attachment of a lipid farnesyl moiety to dependent proteins prior to their insertion into the plasma membrane and thereby prevent activity of these prenylation-dependent proteins, but their mechanism of tumor suppression remains unknown. Many patients receiving FTIs do experience myelosuppression. In this work, the in vitro effects of the FTI, R115777 on normal and leukemic hematopoiesis have been examined as have its effects on apoptosis induction and cell cycle profile in both leukemic blasts and normal CD34+ cells. R115777 was inhibitory to normal CD34+ cell proliferation and to leukemic blast cells, but did not affect long-term culture initiating cell frequency nor NOD-SCID reconstituting capacity. No induction of apoptosis or cell cycle changes were noted in AML blasts. These data suggest that myelosuppression with R115777 occurs largely at the intermediate to late progenitor stage of hematopoiesis and that cyclic use might avoid long-term marrow suppression.

Relationship between human T cell leukemia virus-II and atypical hairy cell leukemia: a serologic study of hairy cell leukemia patients.

Human T cell leukemia virus (HTLV-II) is an infrequently encountered human T cell leukemia virus first isolated from a patient with atypical hairy cell leukemia. Recently, we identified a second patient infected with HTLV-II who had a similar clinical syndrome of atypical hairy cell leukemia associated with peripheral T cell lymphocytosis. HTLV-II was detected by molecular hybridization studies, and more recently, by electron microscopy, in cell lines derived from the patient. Both patients came from the Los Angeles area and had spent several years in Alaska. As opposed to our two patients, 21 patients with more typical cases of hairy cell leukemia were seronegative for HTLV-II. Two additional cases of unusual T cell malignancy linked to HTLV-II have been described by other investigators and bear limited similarity to our index cases. Further studies are necessary to define the spectrum of malignancies linked to HTLV-II and to identify infected individuals for prospective study.

Progressive spastic myelopathy in a patient co-infected with HIV-1 and HTLV-II: autoantibodies to the human homologue of rig in blood and cerebrospinal fluid.

OBJECTIVE: Human T-cell leukemia virus types I (HTLV-I) and II (HTLV-II) are closely related human retroviruses. HTLV-I has been implicated in a chronic progressive myelopathy, known as tropical spastic paraparesis (TSP) or HTLV-I-associated myelopathy (HAM). We sought to determine whether autoantibodies to brain antigens were present in the cerebrospinal fluid (CSF) of a patient with chronic progressive spastic myelopathy with evidence of both HIV-1 infection and HTLV-I/II seropositivity. DESIGN: A 54-year-old bisexual man with clinical features of HAM/TSP of over 20 years' duration was followed. METHODS: We applied discriminatory DNA amplification (polymerase chain reaction) to distinguish HTLV-I from HTLV-II and to verify co-infection with HIV-1. The patient's CSF was used to screen a human brain cDNA expression library to identify antibodies directed against brain antigens. Autoreactive bacteriophage clones were isolated and sequenced. RESULTS: The patient was found to be co-infected with both HIV-1 and HTLV-II, but not with HTLV-I. HTLV-II proviral levels in the peripheral blood remained relatively constant, despite therapy with zidovudine. Prominent oligoclonal banding of immunoglobulins was present in the patient's CSF. A single repeatedly reactive cDNA clone was identified, by screening with CSF antibody, sequenced, and found to be the human homologue of the rat insulinoma gene, rig. CONCLUSIONS: HTLV-II infection may predispose to development of a HAM/TSP-like illness. Autoimmune mechanisms, such as autoantibody formation, may play a role in pathogenesis.

Effects of megakaryocyte growth and development factor on survival and retroviral transduction of T lymphoid progenitor cells.

Murine retroviral vectors have the potential to mediate stable gene transfer into hematopoietic progenitor cells. A known drawback to the use of these vectors is that transduction can only take place in cells actively progressing through the cell cycle. Thrombopoietin, the c-mpl ligand, is known to support division of hematopoietic precursors of primitive origin. Polyethylene glycol (PEG)-conjugated recombinant human megakaryocyte growth and development factor (MGDF) is a polypeptide related to thrombopoietin that stimulates megakaryocyte production. To investigate whether MGDF would also induce stem cell division and support retroviral transduction of CD34+ cells, we compared the effects of MGDF, stem cell factor (SCF), interleukin-3 (IL-3), and IL-6, alone or in combination, using amphotropic and vesicular stomatitis virus (VSV-G) pseudotyped murine retroviral vectors. Similar transduction efficiency was observed when CD34+ cells were transduced in the presence of SCF and MGDF as compared to SCF, IL-3, and IL-6. Using the SCID-hu mouse model of thymopoiesis, we investigated whether CD34+ cells transduced in the presence of these cytokines could reconstitute irradiated thymic implants, and whether vector sequences were present in mature thymocytes. At early timepoints, no significant differences were observed on engraftment of donor progenitors incubated with each cytokine combination. However, a significant difference in the percentage of donor derived CD4+/CD8+ immature thymocytes was observed 9 weeks after implantation of CD34+ cells exposed to the combination of SCF and MGDF as compared to SCF, IL-3, and IL-6 (p = 0.04), indicating that MGDF/SCF better supported the survival of thymocyte precursor cells. Approximately 4% of thymocytes in both cytokine groups harbored vector sequences. These studies provide evidence that MGDF and SCF in combination can mediate transduction of hematopoietic progenitors capable of contributing to long-term thymopoiesis. These results may have important applications for the implementation of gene therapy strategies in disorders affecting the T lymphoid system.

Viral gene delivery to skeletal muscle: insights on maturation-dependent loss of fiber infectivity for adenovirus and herpes simplex type 1 viral vectors.

The mechanisms causing age-dependent loss of muscle fiber infectivity observed in vivo for both adenoviral (Ad) and herpes simplex virus type 1 (HSV-1) gene delivery vectors remain poorly understood. Here we investigate the possible bases for this phenomenon using the novel application of enzymatically isolated, viable, single muscle fibers. We show that maturation-dependent loss of fiber infectivity is recapitulated in single fibers, and, thus, is not solely due to host immune response. Using localized irradiation of muscle in vivo, we show data suggesting that Ad infectivity of differentiated myofibers depends, at least in part, on myoblasts to mediate fiber transduction. On the other hand, infection of single fibers by HSV-1 is not affected by irradiation. Using confocal microscopy, we show that the basal lamina of myogenic cells efficiently infected by HSV-1 is structurally less organized than that of fibers resistant to infection by HSV-1. As well, we show that single myofibers isolated from adult, basal lamina-defective mice (merosin-deficient, dy/dy) are at least 10-fold more susceptible to infection by HSV-1 than are myofibers isolated from control mice. Together, these observations support the hypothesis that the basal lamina acts as a physical barrier to HSV-1 infection of mature muscle.

Human T-cell leukemia viruses: epidemiology, biology, and pathogenesis.

The human T-cell lymphotropic viruses type I and type II are closely related human retroviruses that have similar biological properties, genetic organization and tropism for T lymphocytes. Along with the simian T-cell lymphoma virus type I, they define the group of retroviruses known as the primate T-cell leukemia/lymphoma viruses. Initially identified in 1980, the human T-cell lymphotropic virus type I has been implicated as the etiologic agent of adult T-cell leukemia/lymphoma and of a degenerative neurologic disorder known as tropical spastic paraparesis or human T-cell lymphotropic virus type I-associated myelopathy. The intriguing link between human T-cell lymphotropic virus type, T-cell malignancy, and a totally unrelated and non-overlapping neurological disorder suggests divergent and unique pathogenetic mechanisms. This review will address the epidemiology, molecular biology, and pathogenesis of human T-cell leukemia viruses.

Slow axonal regrowth but extreme hyperinnervation of target muscle after suture of the facial nerve in aged rats.

Unilateral transection and suture of the facial nerve was performed in 60 old rats (20 months of age). The time course of mimetic reinnervation was studied by counting all retrogradely labeled motoneurons in the facial nucleus after injection of HRP into the whiskerpad muscles for 14-112 days post operation. The comparison between these neuron counts and data for young rats yielded four conclusions. First, the qualitative equivalent of the phenomenon "misdirected reinnervation" in aged rats was the same as in young adults: HRP-labeled motoneurons were scattered throughout the facial nucleus lacking myotopic organization from 18 until 112 days post operation. Second, no age-related loss of motoneurons was detected. Third, the axonal regrowth was delayed in aged rats. Fourth, the postoperative hyperinnervation (the projection of more motoneurons into a muscle than under normal conditions, i.e., the quantitative aspect of misdirected reinnervation) was more than two times higher than in young rats. These data may provide reasonable explanations for the poor functional recovery after reconstructive surgery on the facial nerve in old patients.

A patient with progressive myelopathy and antibodies to human T-cell leukemia virus type I and human immunodeficiency virus type 1 in serum and cerebrospinal fluid.

A 52-year-old human immunodeficiency virus type 1-seropositive bisexual black man was evaluated at UCLA because of the recent onset of progressive lower-extremity weakness. Initial neurologic examination showed that the patient's distal weakness was greater than his proximal weakness, with bilateral foot drop and electrophysiologic evidence of denervation in the distal lower extremities. Magnetic resonance imaging of the brain and spinal cord disclosed no abnormalities. Subsequent neurologic evaluation 8 months later showed a myelopathy, with progression of lower-extremity weakness, spasticity, and flexor spasms, and urinary incontinence, as well as the peripheral neuropathy noted previously. A second magnetic resonance imaging scan of the brain showed patchy foci of increased signal intensity in white matter and cortex, with mild generalized cerebral and cerebellar atrophy and no lesions in the spinal cord. Specimens of the patient's serum and cerebrospinal fluid contained antibodies to human immunodeficiency virus type 1. Additionally, specimens of his serum and cerebrospinal fluid were tested for antibody to human T-cell leukemia virus type I by Western blotting and radioimmunoprecipitation, and found to be positive for human T-cell leukemia virus type I gag, env, and tax antibodies. The primary cause of severe myelopathy in this patient may be infection with human T-cell leukemia virus type I rather than with human immunodeficiency virus type 1. Treatment with prednisolone resulted in improvement of the lower-extremity weakness, reduction in flexor spasms, and slower but significant improvement in urinary symptoms. Patients who are infected with human immunodeficiency virus type 1 and have unusual motor findings should be tested for concomitant human T-cell leukemia virus type I infection.

A phase I trial of immunotherapy with intratumoral adenovirus-interferon-gamma (TG1041) in patients with malignant melanoma.

AIMS: Interferon-gamma (IFN-gamma) has been shown to upregulate MHC class I and II expression, and to promote generation of specific antitumor immune responses. We hypothesized that intratumoral administration of an IFN-gamma gene transfer vector facilitates its enhanced local production and may activate effector cells locally. We conducted a phase I dose-escalation study of a replication-deficient adenovirus-interferon-gamma construct (TG1041) to determine safety and tolerability of intratumoral administration, in advanced or locally recurrent melanoma. METHODS: Patients were enrolled at four successive dose levels: 10(7) infectious units (iu) (n=3), 10(8) iu (n=3), 10(9) iu (n=3), and 10(10) iu (n=2) per injection per week for 3 weeks. TG1041 was injected in the same tumor nodule weekly in each patient. Safety, toxicity, local and distant tumor responses and biologic correlates were evaluated. RESULTS: A total of 11 patients were enrolled and received the planned three injections per cycle. One patient with stable disease received a second cycle of treatment. A maximum tolerated dose was not reached in this study. No grade 4 toxicities were observed. Two grade 3 toxicities, fever and deep venous thrombosis were observed in one patient. The most frequently reported toxicities were grade 1 pain and redness at the injected site (n=8), and grade 1 fatigue (n=5) patients. Clinical changes observed at the local injected tumor site included erythema (n=5), a minor decrease in size of the injected lesion (n=5) and significant central necrosis by histopathology (n=1). Systemic effects included stable disease in one patient. Correlative studies did not reveal evidence of immunologic activity. CONCLUSION: Weekly intratumoral administration of TG1041 appears to be safe and well tolerated in patients with advanced melanoma.

Detection of human T-cell leukemia/lymphoma virus type I in a transfusion recipient with chronic myelopathy.

A white man with a progressive spastic paraparesis that began 15 months after sustaining severe trauma in a motor vehicle accident was positive for antibodies to human T-lymphotropic virus type I (HTLV-I) by enzyme-linked immunosorbent assay. Serum antibody to HTLV-I was confirmed by Western blot and radioimmunoprecipitation assay. We detected specific proviral DNA in peripheral blood lymphocytes by the polymerase chain reaction. Because the incidence of HTLV-I is generally restricted to Southern Japan and Caribbean black populations, the most likely source of HTLV-I infection in this patient was multiple intraoperative blood transfusions. The relatively short interval between transfusion and development of HTLV-I-associated myelopathy is consistent with the more rapid evolution of this clinical syndrome compared with adult T-cell leukemia.