Pleiotropic expression of heterologous cytokine/receptor genes in HTLV-1 associated diseases: candidate TRS for chimeric gene therapy.

DNA motifs that encode for specific transcriptional regulatory sequences (TRS) when engineered adjacent to the structural protein coding domain of a suicide enzyme can provide cell-lineage specific protein expression. The disparate up-regulation of several genes in adult T-cell leukemia (ATL) versus HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP), seropositive carriers (SPC) and uninfected normals may reflect events at the molecular level related to leukemogenesis or to processes maintaining the heme-oncologic phenotype. Further, the genetic transduction of cytokine and receptor genes uniquely associated with ATL may provide targets for the development of leukemia-specific gene therapies aimed at exploiting differences in the production of certain growth factors and growth factor receptors. Comparisons of the transcriptional and translational levels of interleukin-2 receptor alpha chain (IL-2R alpha), transforming growth factor-beta 1 (TGF-beta 1) and intracellular adhesion molecule-1 (ICAM-1) in ATL, HAM/TSP, and SPC and in several control populations revealed selectively up-regulated expression in ATL. We evaluated the feasibility of using lymphoid-specific TRS to activate herpes simplex virus thymidine kinase (HSVtk) to achieve selective cytotoxicity in leukemias expressing terminal deoxynucleotidyl transferase (TdT). Selective and efficient leukemic cell killing was produced and suggests that similar chimeric gene constructs containing TRS elements for IL-2R alpha, TGF-beta 1, or ICAM-1 may prove useful in designing gene therapies to treat ATL.

An experimental model of human leukemic meningitis in the nude rat.

An experimental animal model of meningeal leukemia was developed in the nude rat, rnu/rnu, using the human-derived acute lymphoblastic leukemia cell line HPB-ALL. Anesthetized rats were placed in a modified stereotaxic frame and then injected intrathecally, at the level of the cisterna magna, with human leukemic cells. Cerebrospinal fluid and tissue samples from brain, spinal cord, heart, liver, kidney, spleen, bone marrow, and cervical lymph nodes were subjected to histopathologic examination and molecular genetic screening by clonotype primer-directed polymerase chain reaction (CPD-PCR). Ninety-three percent of animals (n = 14) developed signs of meningeal irritation leading to death 30 to 63 days postinjection (median, 36.0 days, mean, 38.7); death occurred between 30 and 39 days in 77% of all animals. Leukemic cells progressively infiltrated the pericerebellar and pericerebral subarachnoid space and infiltrated the Virchow-Robin (perivascular) space. The infiltrating meningeal leukemia closely resembled the pathologic presentation in the human condition. By CPD-PCR, leukemic cells were first detected in cerebrospinal fluid (CSF) on day 4 postinjection, were variably present over the ensuing 17 days, and were consistently detected after day 21. At terminal stages, CPD-PCR tissue surveys showed leukemic DNA in all brains and spinal cords and rarely in cervical lymph nodes, but leukemic DNA was not detected in any other tissue screened. Leukemic meningitis was reliably produced with a predictable survival time. Intrathecal administration of leukemic cells was an efficient means of transmitting leukemic meningitis and it compartmentalized the disease to the central nervous system (CNS), eliminating potential complications of systemic illness. The use of human-derived cell lines may render this model more relevant to the development of future therapeutic strategies to treat leukemia and lymphoma that invade the CNS.

HTLV-associated diseases: human retroviral infection and cutaneous T-cell lymphomas.

An array of neurologic, oncologic, and autoimmune disorders are associated with infection with the human pathogenic retroviruses human T-cell leukemia virus types I and II (HTLV-I, II), as well as the human immunodeficiency viruses (HIV). The cutaneous T-cell lymphomas, mycosis fungoides (MF) and its hematogenous variant Sezary Syndrome (SS), share similar clinical and pathological features to HTLV-I-associated adult T-cell leukemia (ATL) and speculation of a retroviral link to MF and SS, especially in areas non-endemic for ATL, has lead to an intensified search for HTLV- and HIV-like agents in these diseases. To further explore a potential role for human retroviruses in MF and SS, skin biopsy-derived or peripheral blood mononuclear cell-derived DNA from 17 patients (MF, n = 7; erythrodermic MF (EMF), n = 5; SS, n = 5) from the North Eastern United States were screened using gene amplification by PCR and a liquid hybridization detection assay. Previously published primers and probes for HTLV-I (LTR, gag, pol, env, and pX), and our own primers and probes for HTLV-I (gag, pol, and env), HTLV-II (pol and env) and HIV-I (gag and pol) were employed. Serum antibodies to HTLV-I were negative in all but one EMF patient. The single HTLV-I seropositive patient carrying a diagnosis of EMF generated positive amplified signals for all of the eight HTLV-I regions tested. Ultimately, this individual evolved to exhibit clinical manifestations indistinguishable from ATL. The other 16 patients were negative for all 12 HTLV and HIV retroviral regions. Our findings suggest that none of the known prototypic human retroviruses are associated with seronegative MF and SS. The uniformly positive results for HTLV-I in the seropositive patient suggests that this patient initially presented with a smoldering form of ATL and illustrates the difficulty that sometimes may be encountered in the differential diagnosis of MF, SS, and ATL based solely on clinical and histopathological criteria.

Novel compound tetra-, dinucleotide microsatellite polymorphism in the tumor necrosis factor/lymphotoxin locus.

A polymorphic (TGCG)n, tetranucleotide repeat was discovered juxtaposed to the (GT)n dinucleotide repeat that comprises the tumor necrosis factor a microsatellite (TNF) located telomeric to the tumor necrosis factor/lymphotoxin gene cluster. The degree of complexity of this compound tetra-,dinucleotide microsatellite consists of 16 potential alleles of combined length ranging from 24 to 54 bp. The pattern of frequencies of individual alleles belonging to the compound TNFa microsatellite was established from 52 healthy volunteers and was found to be highly heterogeneous. The data diverges significantly from previously published statistics that recognized only a simple variable dinucleotide tandem repeat. The newly recognized compound tetra-, dinucleotide TNFa microsatellite polymorphism establishes a more accurate genetic basis to explore potential linkage with disease susceptibility genes located within this region of the class III major histocompatibility complex. In addition, variable tumor necrosis factor and lymphotoxin production may reflect the more complex polymorphic nature of this microsatellite region. Finally, compound microsatellites probably exist elsewhere, throughout the human genome. Recognition of their presence may have a considerable impact on the validity of past and future microsatellite-based genetic analyses.