Evaluation of macrophage-specific promoters using lentiviral delivery in mice.

In gene therapy, tissue-specific promoters are useful tools to direct transgene expression and improve efficiency and safety. Macrophage-specific promoters (MSPs) have previously been published using different delivery systems. In this study, we evaluated five different MSPs fused with green fluorescent protein (GFP) to delineate the one with highest specificity using lentiviral delivery. We compared three variants of the CD68 promoter (full length, the 343-bp proximal part and the 150-bp proximal part) and two variants (in forward and reverse orientation) of a previously characterized synthetic promoter derived from elements of transcription factor genes. We transduced a number of cell lines and primary cells in vitro. In addition, hematopoietic stem cells were transduced with MSPs and transferred into lethally irradiated recipient mice. Fluorescence activated cell sorting analysis was performed to determine the GFP expression in different cell populations both in vitro and in vivo. We showed that MSPs can efficiently be used for lentiviral gene delivery and that the 150-bp proximal part of the CD68 promoter provides primarily macrophage-specific expression of GFP. We propose that this is the best currently available MSP to use for directing transgene expression to macrophage populations in vivo using lentiviral vectors.

HTLV-1 induced molecular mimicry in neurological disease.

As a model for molecular mimicry, we study patients infected with human T-lymphotropic virus type 1 (HTLV-1) who develop a neurological disease called HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a disease with important biological similarities to multiple sclerosis (MS) (Khan et al. 2001; Levin et al. 1998, 2002a; Levin and Jacobson 1997). The study of HAM/TSP, a disease associated with a known environmental agent (HTLV-1), allows for the direct comparison of the infecting agent with host antigens. Neurological disease in HAM/TSP patients is associated with immune responses to HTLV-1-tax (a regulatory and immunodominant protein) and human histocompatibility leukocyte antigen (HLA) DRB1*0101 (Bangham 2000; Jacobson et al. 1990; Jeffery et al. 1999; Lal 1996). Recently, we showed that HAM/TSP patients make antibodies to heterogeneous nuclear ribonuclear protein A1 (hnRNP A1), a neuron-specific autoantigen (Levin et al. 2002a). Monoclonal antibodies to tax cross-reacted with hnRNP A1, indicating molecular mimicry between the two proteins. Infusion of cross-reactive antibodies with an ex vivo system completely inhibited neuronal firing indicative of their pathogenic nature (Kalume et al. 2004; Levin et al. 2002a). These data demonstrate a clear link between chronic viral infection and autoimmune disease of the central nervous system (CNS) in humans and, we believe, in turn will give insight into the pathogenesis of MS.

Organization of galanin-immunoreactive inputs to the paraventricular nucleus with special reference to their relationship to catecholaminergic afferents.

Immunohistochemical and axonal transport techniques were used to characterize the origin and distribution of galanin-immunoreactive inputs to the paraventricular (PVH) and supraoptic (SO) nuclei of the hypothalamus in the rat. In the parvicellular division of the PVH, the most prominent inputs were confined to the anterior and periventricular parts of the nucleus rostrally and the dorsal and ventral medial subdivisions caudally; the galaninergic inputs to the magnocellular division of PVH and SO were very sparse and were preferentially distributed to regions containing predominantly oxytocinergic neurons. A combined retrograde transport-immunohistochemical method was employed to identify sources of these projections. Galanin immunoreactivity was found to coexist with dopamine-beta-hydroxylase (DBH) immunoreactivity in subsets of retrogradely labeled neurons of the A1 and A6 (locus coeruleus) catecholamine cell groups; no evidence was adduced for the presence of galanin in adrenergic (i.e., phenylethanolamine-N-methyltransferase-positive) neurons that project to the PVH. Apart from minor contributions from the mesencephalic raphe nuclei, no other brainstem cell groups contributed to the galaninergic innervation of the PVH. In the forebrain, the most prominent grouping of doubly labeled cells was centered in the rostral part of the dorsomedial nucleus of the hypothalamus (DMH), though significant numbers were also found in the lateral hypothalamic area, the arcuate nucleus, and the medial preoptic area. In experiments designed to define the subnuclear specificity of some galanin-containing inputs to the PVH, iontophoretic deposits of the anterogradely transported plant lectin, Phaseolus vulgaris-leucoagglutinin (PHA-L), were placed in the A1 and A6 cell groups and in the DMH. Sections through the PVH were prepared so as to allow colocalization of anterogradely transported PHA-L and galanin immunoreactivity in individual fibers and varicosities. Consistent with the retrograde transport data, the greatest degree of galanin-PHA-L correspondence was seen after lectin deposits in the DMH, and over 80% of the doubly labeled varicosities were confined to the anterior, periventricular, and medial parvicellular subdivisions of the nucleus. The galanin-containing projection from the locus coeruleus was most circumscribed, with the vast majority of doubly labeled varicosities confined to the periventricular and adjoining aspects of the anterior and medial parvicellular subdivisions.(ABSTRACT TRUNCATED AT 400 WORDS)

Phase 1 trial of transforming growth factor beta 2 in chronic progressive MS.

Transforming growth factor (TGF)-beta2 is a pleiotropic cytokine associated with remissions in multiple sclerosis (MS) and amelioration of allergic encephalomyelitis. We assessed the safety of TGF-beta2 in an open-label trial of 11 patients with secondary progressive (SP) MS. Five patients had a reversible decline in the glomerular filtration rate. There was no change in expanded disability status scale or MRI lesions during treatment. Systemic TGF-beta2 may be associated with reversible nephrotoxicity, and further investigation of its therapeutic potential in MS should be performed with caution.

Neuropeptide co-expression in the magnocellular neurosecretory system of the female rat: evidence for differential modulation by estrogen.

Single- and double-immunohistochemical staining methods were used to assay the effect of estrogen on the expression of co-existing peptides in the magnocellular neurosecretory system of the female rat. It was confirmed in colchicine-treated, ovariectomized animals that immunoreactive corticotropin-releasing factor and cholecystokinin co-exist in subsets of oxytocinergic neurons; in addition, dynorphin immunoreactivity was detected in a substantial majority of oxytocin containing magnocellular neurons. Consistent with previous studies, magnocellular vasopressinergic cells were found to display angiotensin II-, dynorphin- and galanin-immunoreactivities. Comparable results occurred in colchicine-treated ovariectomized rats independent of whether or not the animals received replacement injections of estradiol benzoate or vehicle. Ovariectomized rats that were not pretreated with colchicine showed enhanced staining (increased cell number and staining intensity of both cell bodies and terminals in the posterior pituitary) for each of the peptides that was found to co-exist in vasopressinergic neurons after treatment with estradiol; staining for vasopressin was similar in steroid- and oil-treated animals. Perikaryal staining for peptides co-localized with oxytocin was not discernibly different in estradiol- vs vehicle-treated animals, while in the posterior lobe, differential effects of hormone replacement on oxytocin, cholecystokinin, and corticotropin-releasing factor immunostaining of terminals were apparent. Perikaryal staining for co-existing peptides in gonadally intact animals killed at the estrus or the diestrus II phases of the estrous cycle provided a pattern of results compatible with those seen in ovariectomized animals treated with estradiol or oil, respectively. These observations suggest that circulating gonadal steroids affect co-existing peptide expression differentially in oxytocinergic vs vasopressinergic neurons. All peptides examined that could be co-localized in vasopressinergic cells showed evidence of enhanced expression in the presence of estrogen, while at least two of these co-localized with oxytocin appeared driven in the opposite direction. The results in normally cycling rats indicate that this kind of influence may be manifest under normal physiological conditions.

Detection of HTLV-I in Peripheral Blood Lymphocytes from Patients with Chronic HTLV-I-Associated Myelopathy/Tropical Spastic Paraparesis and Asymptomatic Carriers by PCR-in situ Hybridization.

Less than 5% of people infected with human T-lymphotropic virus type I (HTLV-I) develop HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic progressive neurologic disease. A number of factors have been implicated in the development of HAM/TSP including heterogeneity of viral sequences, host-genetic background, viral-specific cellular immune responses and viral load. This study examined the presence of HTLV-1 tax DNA in peripheral blood lymphocytes (PBL) from 2 chronic HAM/TSP patients and 2 asymptomatic HTLV-I carriers by using PCR-in situ hybridization (PCR-ISH) for the in situ presence of proviral HTLV-I tax DNA. By this technique, rare PBL from these HTLV-I-infected individuals contained HTLV-I DNA. PCR-ISH did not detect any difference in the number of infected cells between HAM/TSP patients and asymptomatic carriers. Copyright 1997 S. Karger AG, Basel

HTLV-1 and its neurological complications.

BACKGROUND: Since its discovery in 1980, human T-cell lymphotropic virus type-1 (HTLV-1) has been associated with a number of neurological diseases. The distribution of HTLV-1-associated neurological disease is worldwide. In endemic areas, up to 30% of the population may be infected with HTLV-1; however, only a small percentage of infected persons develops neurological disease. REVIEW SUMMARY: In 1986, HTLV-1 infection was reported in patients of chronic progressive myelopathy of uncertain etiology, and the disease entity was called HTLV-1-associated myelopathy/tropical spastic paraparesis. Recently, HTLV-1 infection has been associated with polymyositis and uveitis. Interestingly, a single patient may display more than one syndrome. Although other neurological syndromes occur in HTLV-1-infected individuals, there is not enough epidemiologic data that show a strong association. Treatment of HTLV-1-associated neurological disease is challenging, and well-controlled studies are lacking. CONCLUSION: As neurologists and other scientists begin to understand the pathophysiology of HTLV-1 infection, improved therapies should be developed. Randomized trials with longer follow-up are required to understand the effect of treatment on disability and quality of life.

Molecular mimicry in neurological disease: what is the evidence?

Autoimmune diseases are a leading cause of disability and are increasing in incidence in industrialized countries. How people develop autoimmune diseases is not completely understood, but is related to an interaction between genetic background, environmental agents, autoantigens and the immune response. Molecular mimicry continues to be an important hypothesis that explains how an infection with an environmental agent results in autoimmune disease of the nervous system and other target organs. Although molecular mimicry has yet to be unequivocally proven, in the past several years there has been a sharpening of its definition with better experimental data implicating it as a cause of neurological disease in humans.

HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP): a chronic progressive neurologic disease associated with immunologically mediated damage to the central nervous system.

This review examines information on clinical, pathological and immunological events in the slowly progressive neurologic disorder associated with the human T lymphotropic virus type-I (HTLV-I) termed HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP). The role of cellular immune responses to HTLV-I in patients with HAM/TSP and how these responses may be associated with the pathogenesis of this disorder will be discussed. While a number of immunologic responses have been shown to be abnormal in HAM/TSP patients, studies on HTLV-I specific cytotoxic T cell responses (CTL) will be specifically examined. By defining such antigen specific functional cellular host responses to HTLV-I we hope to better understand the underlying mechanisms that may be involved in the neuropathology of HTLV-I associated neurologic disease. This has led to a number of HTLV-I associated immunopathogenic models that may be operative in HAM/TSP patients. Importantly, based on these models, potential immunotherapeutic strategies for disease intervention can be devised. Moreover, such an analysis may have significant implications for our understanding of other HTLV-I associated clinical disorders and other neurological diseases in which viral etiologies have been suggested.

Estrone sulfate stimulates growth of nitrosomethylurea-induced breast carcinoma in vivo in the rat.

Concentrations of estradiol in human breast tumors from pre- and post-menopausal women are similar whereas plasma levels are 5- to 60-fold lower in post-menopausal women. The mechanism for maintaining high tumor tissue estrogen levels in post-menopausal women is unknown but could be related to the ability of plasma estrone sulfate to serve as a precursor for estradiol synthesis in tumor tissue. Estrone sulfate plasma levels are 30-fold higher than free estradiol levels in post-menopausal women and estrone sulfatase is present in many tissues, including breast tumors, supporting this hypothesis. In this study, we examined the ability of exogenously administered estrone sulfate to stimulate growth of a carcinogen-induced, hormone-dependent rat mammary tumor and measured the rate of conversion of estrone sulfate to free estrone and estradiol. Castrate rats bearing nitrosomethylurea-induced mammary tumors were infused with estradiol as a control or estrone sulfate over a 14-day period. Estradiol at low doses significantly increased tumor volume whereas higher amounts paradoxically inhibited growth. By comparison, estrone sulfate infusions significantly increased tumor volume over that observed in castrate animals on both days 7 and 14 of infusion. To determine whether estrone sulfate was converted to free estrone and estradiol during this protocol, 3H-estrone sulfate was substituted for unlabelled steroid and castrate animals were again infused for 14 days. At 7, 10 and 14 days of infusion, 18-26% of estrone sulfate was converted to free estrone and 9-16% to free estradiol. There were no significant differences between the 2 doses used and the rates of conversion were stable over the infusion period. Conversion of estrone sulfate to free estradiol was also demonstrated by radioimmunoassay of free estradiol in plasma during estrone sulfate infusions. These data demonstrate that exogenously administered estrone sulfate can stimulate mammary tumor growth in castrate animals and support the possibility that estrone sulfate may serve as an important source of tumor tissue estradiol.

Tumor necrosis factor alpha expression in the spinal cord of human T-cell lymphotrophic virus type I associated myelopathy/tropical spastic paraparesis patients.

HTLV-I Associated Myelopathy Tropical Spastic Paraparesis (HAM/TSP) is a chronic degenerative disease mainly affecting the spinal cord. The pathogenesis of HAM/TSP is unknown, but is thought to involve immunopathogenic mechanisms. Several reports have detected inflammatory cytokines such as tumor necrosis factor alpha (TNF alpha) in HAM/TSP patients. In this study, we used in situ hybridization (ISH) to examine the expression of TNF alpha RNA in spinal cord autopsy specimens from three chronic HAM/TSP patients with long-term disease (10-20 years duration). ISH identified many TNF alpha-expressing cells throughout all three patient's spinal cord tissues. Two patient's spinal cord tissue showed inflammatory cells, however double labeling by ISH for TNF alpha RNA with immunohisto-chemistry for CD45RO (a marker for memory T-cells) or CD-68 (a marker for microglia/macrophages) did not colocalize TNF alpha RNA with either CD45RO or CD-68 positive cells. Therefore, TNF alpha is expressed in the spinal cord of chronic HAM/TSP patients compared to normal controls and TNF alpha-expressing cells do not appear to be memory T-cells, microglia or macrophages.

Localization of retrovirus in the central nervous system of a patient co-infected with HTLV-1 and HIV with HAM/TSP and HIV-associated dementia.

Persons co-infected with HTLV-1 and HIV are at increased risk for neurologic disease. These patients may develop HAM/TSP and/or HIV-associated dementia. In this study, we localized cells infected with retrovirus in the central nervous system (CNS) of a patient with both HAM/TSP and HIV-associated dementia. HTLV-1 was localized to astrocytes and HIV to macrophage/microglia. There was no co-infection of a single cell phenotype in this patient. These data suggest that mechanisms other than co-infection of the same CNS cell may play a role in the development of neurologic disease in patients dual infected with HTLV-1 and HIV.

IgG in brain correlates with clinicopathological damage in HTLV-1 associated neurologic disease.

OBJECTIVE: To test the authors' hypothesis that antibody deposition in autopsy specimens from patients with human T-lymphotropic virus type 1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) would correlate with CNS damage. METHODS: Endogenous immunoglobulin G (IgG) was detected using antihuman IgG in autopsy tissues from HAM/TSP and control patients. IgG was isolated from the CSF, CNS, and sera of patients with HAM/TSP and tested for reactivity to heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), an autoantigen recently associated with molecular mimicry in HAM/TSP. RESULTS: In situ IgG localized to elements of the corticospinal system including neurons of the frontal cortex and precentral gyrus, as well as throughout axons in subcortical white matter, periventricular white matter, posterior limb of the internal capsule, midbrain, pons, and medulla. Similarly, there was IgG deposition within the posterior-column/medial lemniscal sensory system, including the arcuate fibers of the cranial-cervical junction, the nucleus cuneatus, and throughout the course of the medial lemniscus in the medulla, pons, and midbrain. IgG from brain, CSF, and serum of the patients with HAM/TSP showed immunoreactivity with hnRNP A1. CONCLUSION: Patients with HAM/TSP develop antibodies specific for neurons and axons that are preferentially damaged in the CNS.

PCR-in situ hybridization detection of human T-cell lymphotropic virus type 1 (HTLV-1) tax proviral DNA in peripheral blood lymphocytes of patients with HTLV-1-associated neurologic disease.

PCR-in situ hybridization (PCR-ISH) was developed and utilized to determine the distribution of human T-cell lymphotropic virus type 1 (HTLV-1) tax proviral DNA in peripheral blood lymphocytes (PBL) from patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). PCR-ISH of HTLV-1 tax DNA in PBL from patients with HAM/TSP revealed that 1 in 5,000 to 1 in 10,000 PBL contained virus. PCR-ISH was sensitive, because a positive signal was consistently demonstrated from the HTLV-1-infected cell lines HUT-102 (which contains four to six copies of HTLV-1 proviral DNA per cell) and MT-1 (which contains one to three copies of HTLV-1 proviral DNA per cell). Also, intracellular amplification by PCR-ISH significantly increased sensitivity compared with conventional ISH and was shown to be specific for HTLV-1 tax DNA. These results are in contrast to solution-phase PCR amplification in which greater than 1% of cells were estimated to be infected. The discordance between these results is discussed and may indicate that more than one copy of HTLV-1 tax proviral DNA is present in an individual PBL.

Detection of human T-lymphotropic virus type I (HTLV-I) tax RNA in the central nervous system of HTLV-I-associated myelopathy/tropical spastic paraparesis patients by in situ hybridization.

Autopsy specimens from 3 patients with human T-lymphotropic virus (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) were examined for the presence of HTLV-I in the central nervous system (CNS). In situ hybridization using an HTLV-I tax RNA probe detected cells containing HTLV-I RNA in spinal cord and cerebellar sections. HTLV-I infected cells were located within the white matter and, in particular, within the anterior and lateral funiculi of the spinal cord. Consistent with previously described HAM/TSP pathology, there were perivascular infiltrates in these CNS specimens. Significantly, HTLV-I RNA was not localized to these infiltrates but was detected deeper within the neural tissue. Furthermore, phenotypic analysis demonstrated that at least some of the infected cells were astrocytes. While previous polymerase chain reaction studies have demonstrated the presence of proviral HTLV-I in CNS specimens, here we provide evidence for the in situ expression of HTLV-I RNA in the CNS of HAM/TSP patients.

Neuronal molecular mimicry in immune-mediated neurologic disease.

Molecular mimicry is implicated in the pathogenesis of autoimmune diseases such as diabetes mellitus, rheumatoid arthritis, and multiple sclerosis (MS). Cellular and antibody-mediated immune responses to shared viral-host antigens have been associated with the development of disease in these patients. Patients infected with human T-lymphotropic virus type I (HTLV-I) develop HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), an immune-mediated disorder of the central nervous system (CNS) that resembles some forms of MS. Damage to neuronal processes in the CNS of HAM/TSP patients is associated with an activated cellular and antibody-mediated immune response. In this study, IgG isolated from HAM/TSP patients was immunoreactive with uninfected neurons and this reactivity was HTLV-I specific. HAM/TSP IgG stained uninfected neurons in human CNS and cell lines but not nonneuronal cells. Neuronal western blots showed IgG reactivity with a single 33-kd band in all HAM/TSP patients tested. By contrast, no neuron-specific IgG reactivity could be demonstrated from HTLV-I seronegative controls and, more important, from HTLV-I seropositive, neurologically asymptomatic individuals. Both immunocytochemical staining and western blot reactivity were abolished by preincubating HAM/TSP IgG with HTLV-I protein lysate but not by control proteins. Staining of CNS tissue by a monoclonal antibody to HTLV-I tax (an immunodominant HTLV-I antigen) mimicked HAM/TSP IgG immunoreactivity. There was no staining by control antibodies. Absorption of HAM/TSP IgG with recombinant HTLV-I tax protein or preincubation of CNS tissue with the monoclonal antibody to HTLV-I tax abrogated the immunocytochemical and western blot reactivity of HAM/TSP IgG. Furthermore, in situ human IgG localized to neurons in HAM/TSP brain but not in normal brain. These data indicate that HAM/TSP patients develop an antibody response that targets uninfected neurons, yet reactivity is blocked by HTLV-I, suggesting viral-specific autoimmune reactivity to the CNS, the damaged target organ in HAM/TSP.

HTLV-I/II seroindeterminate Western blot reactivity in a cohort of patients with neurological disease.

The human T-cell lymphotropic virus type I (HTLV-I) is associated with a chronic, progressive neurological disease known as HTLV-I-associated myelopathy/tropical spastic paraparesis. Screening for HTLV-I involves the detection of virus-specific serum antibodies by EIA and confirmation by Western blot. HTLV-I/II seroindeterminate Western blot patterns have been described worldwide. However, the significance of this blot pattern is unclear. We identified 8 patients with neurological disease and an HTLV-I/II seroindeterminate Western blot pattern, none of whom demonstrated increased spontaneous proliferation and HTLV-I-specific cytotoxic T lymphocyte activity. However, HTLV-I tax sequence was amplified from the peripheral blood lymphocytes of 4 of them. These data suggest that patients with chronic progressive neurological disease and HTLV-I/II Western blot seroindeterminate reactivity may harbor either defective HTLV-I, novel retrovirus with partial homology to HTLV-I, or HTLV-I in low copy number.