Cell Line Hut78 Research Articles

Tumor necrosis factor alpha selectively sensitizes human immunodeficiency virus-infected cells to heat and radiation.

We report here that infection of the human T-cell line HUT-78 with human immunodeficiency virus (HIV) increases its sensitivity to heat and radiation toxicity. A possible explanation for this result may be the reduced expression of manganous superoxide dismutase (MnSOD) in HIV-infected cells compared to uninfected cells. Tumor necrosis factor alpha (TNF-alpha) further sensitizes HIV-infected cells but not uninfected cells to heat and radiation. This is consistent with the ability of TNF-alpha to induce the expression of MnSOD in uninfected but not in HIV-infected cells. HIV-infected HUT-78 cell lines engineered to overexpress MnSOD are more resistant to heat and radiation than HIV-infected cells that do not overexpress MnSOD. However, treatment with TNF-alpha still sensitizes these cells to heat and radiation.

Cytokine enhancement of simian immunodeficiency virus (SIV/mac) from a chronically infected cloned T-cell line (HuT-78)

Simian immunodeficiency viruses (SIV) are a family of primate lentiviruses similar to human immunodeficiency viruses (HIV) in their genetic sequence and pathogenesis. However, host-derived cofactors which may determine the extent of viral replication are not clearly defined for SIV or HIV infections. A HuT-78 cell line chronically infected with SIV/mac strain 251, was biologically cloned and characterized for the ability to produce infectious viral particles, viral structural protein profile, cellular antigen surface phenotype and tested to determine the effects of recombinant cytokines on SIV replication. Reverse transcriptase (RT) assay was used to measure the replication of SIV/mac in response to various concentrations of recombinant cytokines (1-1000 units/ml). We report that tumor necrosis factor-alpha (rTNF-alpha), gamma-interferon (rIFN-gamma), interleukin 2 (rIL-2), and granulocyte-macrophage colony stimulating factor (rGM-CSF) induced approximately a 2 to 3 fold increase in virus RT activity compared with untreated SIV-infected HuT-78 cells. In contrast, viral replication was not enhanced or minimally enhanced by interleukin 1 (rIL-1), interleukin 3 (rIL-3), or interleukin 4 (rIL-4) at similar dosages. Furthermore, SIV replication in response to rTNF-alpha and rIFN-gamma occurred in a dose dependent fashion. These data suggest that SIV-infected T-lymphocyte lines are responsive to particular cytokines resulting in increased virus production.

Biological characterization of infectious molecular clones derived from a human immunodeficiency virus type-1 isolate with rapid/high replicative capacity

In order to molecularly characterize rapidly and slowly replicating HIV-1 variants, molecular clones were obtained from a rapid/high virus isolate. This isolate, 4803, had only been passaged in peripheral blood mononuclear cells (PBMC) prior to cloning. Molecular cloning was done in bacteriophage λ-dash using high molecular weight DNA of isolate 4803 infected PBMC. Seven recombinant phages were identified. The clones were found to be related to each other and differed only at 1 or 2 restriction sites (out of 28). The molecular clones were transfected into various cell types by electroporation. The phenotype of progeny viruses was found to be dependent on the cell type used for transfection. Progeny viruses produced by PBMC cultures differed from the parental isolate in that they did not form syncytia and lacked the capacity to replicate in cell lines. Since transfection of PBMC yielded progeny viruses within 1 week, this phenotype is considered to be the true phenotype of the clones. Transfection of the T-lymphoid HUT-78 cell line and of the monocytoid U937-2 cell line yielded progeny viruses after considerable delay (more than 1 month). Progeny viruses from HUT-78 cells were similar to the parental isolate in that they formed syncytia in PBMC and replicated in all cell lines tested. Progeny viruses from U937-2 cells showed an intermediate phenotype in that they replicated in U937-2 but not in T-lymphoid cell lines. These results indicate that molecular clones of a rapid/high virus may have a restricted replicative capacity compared to the parental, genetically heterogenous virus isolate.

Detection of human T-cell leukemia virus type I (HTLV-I) provirus in an infected cell line and in peripheral mononuclear cells of blood donors by the nested double polymerase chain reaction method: comparison with HTLV-I antibody tests

Human T-cell leukemia virus type I (HTLV-I) provirus DNA from the cultured cell line HUT 102 and from peripheral mononuclear cells (PBMC) of anti-HTLV-I antibody-positive Japanese blood donors was detected by the nested double polymerase chain reaction (PCR) method. This procedure consists of a first amplification and a second amplification with the products of the first amplification and primers interior to the first primers. Using this method, we demonstrated that it is possible to detect single-template DNA. Polyacrylamide gel electrophoresis of the nested double PCR products, with our primers, revealed three bands with excess amounts of template DNA, two bands with moderate amounts, and a single band with limited amounts. The amount of provirus in PBMC was roughly estimated from the results of the nested double PCR. Particle agglutination (PA) assays and indirect immunofluorescence testing (IF) with mixed MT-2 cells and Molt-4 cells as targets to detect anti-HTLV-I antibody were performed, and the results were compared with those of the nested double PCR of the pX region. None of the 101 PA-negative samples were positive in either the IF or PCR test. Of the 155 samples that were antibody positive by the PA assay, 57 were positive by both PCR and IF. Furthermore, the results of the IF and PCR tests coincided completely. It was therefore concluded that the IF method is most appropriate for confirmation of the PA assay currently used in most diagnostic laboratories and blood centers.

Kinetic studies and unstructured models of lymphocyte metabolism in fed‐batch culture

The growth of two lymphocyte cell lines, a hybridoma cell line and a human cutaneous T cell lymphoma (HuT78), was studied in fed-batch culture, and unstructured models of growth developed. A criteria was established to insure that the growth rate varied by less than a specified tolerance throughout the culture period. Glutamine and serum were growth-limiting nutrients for both cell lines with half-maximal growth rates at 0. 53 mM glutamine and 0. 55%(v/v) serum for the hybridoma cells and 0. 21 mM glutamine and 1. 5% serum for the HuT-78 cells. Over the range of glucose concentrations from 5. 5 mM to 28 mM, the specific growth rate of hybridoma cells was independent of glucose concentration, whereas glucose concentrations above 5. 5 mM inhibited HuT-78 growth. For both cell lines, the growth rate was significantly inhibited by the addition of ammonium, although the hybridoma cell line was more affected by ammonia than was the HuT-78 cell line. Growth of HuT-78 cells increased in the presence of interleukin-2. Unstructured models for the hybridoma cells were similar to other models presented in the literature. Applications of these models to adoptive immunotherapy are discussed.

A specific defect of prosolin phosphorylation in T cell leukemic lymphoblasts is associated with impaired down-regulation of DNA synthesis.

Prosolin is a major cytosolic phosphoprotein of proliferating normal PBL. Treatment of growing PBL with phorbol ester (12-O-tetradecanoylphorbol-13-acetate (TPA)) or calcium ionophore (A23187) for 1 h caused phosphorylation of prosolin with the production of up to four prominent phosphorylated forms differing in degree of phosphorylation and/or two-dimensional electrophoretic mobility (peptides B to E). Formation of these phosphopeptides coincided with rapid down-regulation of DNA synthesis. A23187 was particularly effective in inducing phosphorylation of the more highly phosphorylated peptides D and E, suggesting the existence of a (Ca2+)-activated mechanism in their phosphorylation. The T cell leukemia cell lines Jurkat, HuT-78, CCRF-CEM, and Molt-4 showed reduced to absent ability to phosphorylate prosolin peptides rapidly in response to A23187 and also showed diminished down-regulation of DNA synthesis. In leukemic cells treated with both TPA and A23187, peptides B and C were rapidly phosphorylated, but the phosphorylation of peptides D and E seen in normal PBL remained deficient. The T cell leukemic cells appear to have intact a TPA-activated mechanism for phosphorylating prosolin peptides B and C, but share an impairment of a specific Ca2(+)-activated mechanism, possibly a Ca2(+)-dependent protein kinase, required for phosphorylation of prosolin phosphopeptides D and E. The degree of rapid down-regulation of DNA synthesis was correlated with degree of phosphorylation of peptide E in PBL and in three of four T cell leukemic cell lines. Thus, rapid phosphorylation of prosolin may mediate responses to TPA and A23187 in normal proliferating PBL, including down-regulation of DNA synthesis. A deficiency of this pathway in leukemic T cells may impede their response to physiologic growth regulatory signals utilizing this pathway and contribute to unrestrained cell growth.

Identification of effector cell protease receptor-1. A leukocyte-distributed receptor for the serine protease factor Xa.

Mitogenesis, cell differentiation and immune-inflammatory responses are regulated by the coordinated assembly of proteases with specific cellular receptors. We have investigated the possibility that immune effector cells may express a high-affinity protease receptor. To address this hypothesis, we have generated mAb to factor V and its activated form Va, a circulating plasma protein that binds the serine protease of the coagulation cascade, factor Xa. Further, by flow microfluorimetry screening, we have isolated a panel of these mAb that recognize a surface molecule expressed on transformed monocytic cells. We now show that these mAb bind to blood monocytes, to CD3- CD16+ CD56+ NK cells, and with considerable heterogeneity, to neutrophils. A small subset of CD3+ cells (5 to 10%) was also identified by these probes and further phenotypically characterized by two-color flow microfluorimetry as predominantly coexpressing CD2, CD4 or CD8, CD57, CD11b, and alpha/beta TCR. This subset of CD3+ cells was expanded in vitro by both lectin- or Ag-specific stimulation. In addition, long term alloreactive stimulation resulted in approximately 8- to 10-fold increased expression of the molecule recognized by these mAb. Functional analyses were performed on a selected T cell clonal derivative of the transformed cell line HuT 78. These cells bound 125I-factor Xa in a specific reaction saturated at 194,000 +/- 26,000 molecules/cell with a Kd approximately 10 to 20 nM and inhibited by the mAb panel described above. These data suggest that immune effector cells express a dynamically regulated protease receptor that is immunologically related to the plasma coagulation protein factor V and its activated form Va. We propose the term effector cell protease receptor-1 to tentatively identify this molecule, and we speculate on its possible involvement in specialized protease-mediated effector functions.

Modulation of the Interleukin 2 Receptor by Maternal and Cord Blood Serum

In this report, the potential inhibitive action, of maternal serum (retroplacental and peripheral), and cord blood serum on the expression of the Il2r has been studied. Calf and male serum were used as a control. In order to have an optimal Il2r expression, a previous PHA cellular stimulation was performed. The maternal serum's Il2r inhibitive property was measured during and after the pregnant period. The presence of Il2r on maternal lymphocytes, cord blood cells, and unrelated donor mononuclear cells has been investigated (after a PHA stimulation assay). The downregulated Il2r expression of neoplastic cell line (Hut78 cell line) under the influence of maternal serum has been observed. The examination of the inhibitive action due to maternal serum has suggested that a factor included in the IgG fraction is mainly responsible for the downregulating property concerning the Il2r expression. A possible mechanism for the action of this factor has been studied. Further experiments suggest that the addition of recombinant Il2 during the action of low doses of maternal IgG allows a partial reexpression of the Il2 receptor. However, at physiological concentrations of IgG, the Interleukin 2 receptor downregulation becomes irreversible.

Cotrimoxazole in acute uncomplicated urinary tract infections in women

Arsenic trioxide (As2O3) displays apoptogenic properties against various types of hematopoietic malignancies. We investigated the effects of As2O3 on the viability of the cutaneous T cell lymphoma cell lines HuT-78, SeAx, and Myla, and of peripheral blood mononuclear cells from patients with Sézary syndrome, by using propidium iodide and annexin-V staining, terminal deoxyuridine triphosphate nick end labeling (TUNEL), cell cycle analysis, mitochondrial transmembrane potential (ΔΨm) alterations, cytochrome c release, and detection of processed caspase-3. We also report in vivo effects of As2O3 in two patients with cutaneous T cell lymphoma. The results show that As2O3 induces apoptosis of cutaneous T cell lymphoma lines and of Sézary cells from patients in a time- and concentration-dependent manner in vitro, as demonstrated by annexin-V staining, mitochondrial depolarization, and DNA fragmentation. Ascorbic acid 100 μM potentiated As2O3-induced Sézary cell death, whereas interferon-α had no synergistic effect. As2O3-induced Sézary cell death involves activation of caspase-3, cleavage of poly(ADP-ribose)polymerase, and cytochrome c release, but was only partially inhibited by the pan-caspase inhibitor Z-VAD.fluoromethylketone. Finally, As2O3 was administered to two patients with cutaneous T cell lymphoma, allowing us to obtain a partial response in one case, whereas stability was observed in the second patient. These results demonstrate that As2O3 synergizes with ascorbic acid to induce Sézary cell death at clinically achievable concentrations, through a caspase-partially independent pathway, and provide a rationale for further in vivo studies addressing the therapeutic efficacy of As2O3 in cutaneous T cell lymphoma patients.

Lymphotoxin cDNA clones from a HTLV-I-carrying T cell line HUT-102.

The conditioned medium of a HTLV-I-carrying T cell line HUT-102 showed cytotoxic activity against a mouse fibroblast cell line L-M. We prepared the cDNA library from HUT-102 poly(A)+ RNA and screened it using oligonucleotide probes that correspond to the amino acid sequences conserved in tumor necrosis factor (TNF) and lymphotoxin (LT). As a result we obtained two kinds of cDNA clones encoding LT in which amino acid residue 26t of mature LT is different; one is Asn and another is Thr. The sequence of the genomic clones obtained using a polymerase chain reaction method showed that the HUT-102 genome also contains two types of LT genes. Recombinant LTs expressed in Escherichia coli exhibited the same level of cytolytic activity against L-M cells. These results indicate that the cytotoxin constitutively produced by HUT-102 cells include two kinds of LT.

The 75,000-Dalton Interleukin-2 Receptor Transmits a Signal for the Activation of a Tyrosine Protein Kinase

The high-affinity receptor for interleukin-2 (IL-2) is composed of two distinct subunits with molecular weights of 55,000 and 75,000 (p55 and p75). While the presence of the high-affinity receptor requires the simultaneous expression of p55 and p75, these subunits can also be expressed independently, resulting in IL-2 receptors with low and intermediate affinities, respectively. IL-2 can induce proliferation in cells expressing either the intermediate affinity p75 receptor or the p55.p75 high-affinity complex, suggesting that p75 is responsible for signal transduction. We have previously shown that signal transduction by the high-affinity IL-2 receptor involves the activation of a tyrosine protein kinase. In order to evaluate the role of p75 in the activation of this kinase we assessed the ability of IL-2 to induce the activation of a tyrosine protein kinase in the human leukemic cell lines Hut 78 and YT. These cells express p75 as the predominant IL-2 receptor. IL-2-dependent tyrosine phosphorylation was observed in both cell lines and the concentrations of IL-2 needed to stimulate this phosphorylation were similar to that required for binding to the p75 receptor. Antibodies that inhibit binding of IL-2 to p55 had no effect on the IL-2-induced tyrosine phosphorylations in YT cells, while antibodies that block the binding of IL-2 to p75 completely inhibited the phosphorylations. These results demonstrate that the signaling capacity for the IL-2-induced tyrosine phosphorylation resides in the p75 receptor.

Molecular and biological characterization of a replication competent human immunodeficiency type 2 (HIV-2) proviral clone.

We obtained complete genomic clones of human immunodeficiency virus type 2 (HIV-2) from the DNA of the neoplastic human cell line HUT 78 freshly infected with a HIV-2 isolate, strain SBL6669. The recombinant phage DNA was transfected into the lymphocytes of CD4-positive HUT 78 cell line to test the replication competence of the proviral DNA. One genomic clone, designated HIV-2SBL/ISY, yielded retroviral particles after a few weeks of culture of the transfected cells. The HIV-2SBL/ISY clone contained a complete provirus and cellular flanking sequence. We obtained the DNA sequence of the provirus and compared it with the published sequence of two other HIV-2 isolates. The degree of variability among HIV-2 isolates is comparable to that observed among African HIV-1 isolates sequenced to date. Immunologically, HIV-2SBL/ISY is similar to the parental virus (HIV-2SBL6669) but differs in the envelope transmembrane protein that is truncated (gp32-34) in the parental virus and not in HIV-2SBL/ISY (gp41). Both the parental and the cloned viruses are infectious and cytopathic for some human T-cell lines, induce syncytia, and infect a human macrophage cell line (U937) in vitro. The availability of a biologically active HIV-2 clone provides the means to study the role and interaction of HIV-2 genes in vitro as well as to assess the functional similarities among HIV-1 and HIV-2 genes. Since HIV-2SBL/ISY cloned virus infects fresh peripheral blood T cells from Rhesus macaques in vitro and infects the same animal in vivo, its use in animals may represent a model for functional study of viral genes in vivo as well as for development of experimental approaches to prevent and cure retroviral infection in humans.

Processing and Secretion of Envelope Glycoproteins of Human Immunodeficiency Virus Type 1 in the Presence of Trimming Glucosidase Inhibitor Deoxynojirimycin

The processing and secretion of the envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) were studied in chronically infected cells treated with the trimming glucosidase inhibitor deoxynojirimycin (DNM). In Molt3 cells infected with human T-lymphotropic virus type III (HTLV-IIIB), DNM inhibited the intracellular proteolytic processing of gp160 to gp120 and gp41. A clone of the HUT78 cell line called 6D5, when chronically infected with the HIV-1 isolate HTLV-III451 was shown to release both gp160 and gp120 into the culture medium. The secretion of envelope glycoproteins from these infected cells was not inhibited by DNM treatment. The secreted proteins had higher molecular weights than gp160 and gp120 from cultures not treated with DNM, presumably due to the presence of unprocessed carbohydrate residues on the polypeptide chain. These secreted glycoproteins from DNM-treated cells exhibited specific interaction with the CD4 molecule on the surface of target cells. However, the syncytium formation induced by HIV-1-infected cells on CD4+ cells was significantly inhibited in the presence of the glucosidase inhibitor. The minimal cytotoxicity of the DNM coupled with its strong inhibitory effect on the cell-to-cell spread of the virus suggest that it may be potentially useful in antiviral drug therapy of HIV-1 infection.

Expression of a leukemia-associated antigen (CAMAL) in four myeloid leukemia cell lines

We have previously described the detection and partial characterization of a common myelogenous leukemia-associated antigen (CAMAL), in CGL and ANLL patients. Both polyclonal and monoclonal (CAMAL-1) antibodies have been raised to p70 (CAMAL) and have been shown to react with both p70 and myeloid leukemia cell preparations. p70 (CAMAL) has been shown to be a monomeric protein of M r 70,000 and pI 7.2 and was also detectable in the myeloid leukemia cell lines HL60, KG1, K562 and U937, but not in the lymphocytic cell lines Molt-4, Hut-78 and CEM by immunoprecipitation from iodinated cell samples. Using [ 35S] methionine-labeled cell lines and immunoprecipitation, we have demonstrated the constitutive expression of p70 as well as a major component at p58 and a number at lower molecular weights in the myeloid leukemia cell lines HL60, KG1, K562 and U937, but not in the lymphocytic leukemia cell lines Molt-4, Hut-78 and CEM. The implications of these observations are discussed.

A unique human immunodeficiency virus culture secreting soluble gp160.

A clone of the HUT78 cell line, chronically infected with the HIV-1 isolate HTLV-III451, has been demonstrated to secrete unprocessed HIV-1 envelope precursor protein gp160 as well as mature gp120. Further, when grown in serum-free defined medium these cells released approximately five times the amount of virus compared with cultures in normal medium. These proteins corresponded in their immunologic reactivities with the respective envelope proteins of the HTLV-IIIB isolate. They formed high-affinity soluble complexes with the CD4 antigen and inhibited the syncytium formation induced by HTLV-IIIB on CD4-positive cells. This is the first description of an HIV-1 culture system capable of shedding into the medium native gp160 that is soluble in the absence of detergents.

Long-term persistent infection of macaque monkeys with the simian immunodeficiency virus.

Juvenile rhesus macaques 6 to 18 months of age were experimentally infected by intravenous inoculation with the simian immunodeficiency virus (SIV), the T cell-tropic retrovirus of monkeys related to the human acquired immunodeficiency syndrome (AIDS) virus HIV. The SIV used for inoculation was grown either in normal human peripheral blood lymphocytes in the presence of interleukin 2 or in the human tumour cell line HUT-78. Eight of the macaques died 129 to 352 days post-inoculation with a variety of clinical and pathological findings paralleling those of AIDS in humans. However eight other animals became persistently infected for prolonged periods; these eight macaques remained alive at 537 and 820 days post-inoculation despite persistent lymphadenopathy and our continued ability to isolate SIV. The ability of these monkeys to survive infection correlated directly with the strength of their antibody response to SIV. Infection was also established in macaques using approximately 100 tissue culture infectious doses of HUT-78-grown SIV. There was no correlation between the dose of virus inoculum and either the strength of the antibody response or clinical outcome. These results demonstrate that SIV infection of macaques can be used not only to study acute AIDS but also to mimic the long-term persistent infection seen in carriers of HIV.

Differential ability of human immunodeficiency virus isolates to productively infect human cells.

Isolates of HIV showed distinct differences in the ability to replicate in continuous human hematopoietic cell lines. Moreover, although all PMC cultures obtained from healthy individuals could be infected with HIV, considerable variation in the amount of virus released from different PMC cultures was observed. These biological properties of HIV could not be correlated with clinical state, binding properties of the virus isolates to target cells, or differences in target cell CD4 antigen expression. Some isolates of HIV that could not directly infect the HUT-78 cell line showed productive infection when PMC infected with these viruses were added to this human T cell line. These observations emphasize the importance of cell to cell contact in the spread of virus. The results demonstrate for the first time the differences in the host range specificity of HIV isolates in several individual PMC cultures, and indicate that the optimal isolation of HIV is achieved with normal human PMC rather than established human cell lines.

A monoclonal antibody (FMG25) that can differentiate neuroblastoma from other small round-cell tumours of childhood.

The monoclonal antibody (MAb), FMG25, raised following immunization of mice with the human T cell line HUT 78, binds to human neuroblastoma but not to other small round-cell tumours of childhood (rhabdomyosarcoma and Ewing's sarcoma). The specificity of the reagent is paralleled on normal tissues binding to brain but not frozen sections of thymus, tonsil, lymph node and spleen. Of the 15 T-cell malignancies examined, only 3, belonging to the large T-cell type, were positive. This represents only 50% of this type examined. Only one early case of pre-B ALL was found to be positive for FMG25 binding out of 27 non-T-cell malignancies. The pattern of reactivity of FMG25 makes the MAb useful as a diagnostic reagent in paediatric pathology. In addition, the antibody may be useful as one of a panel of reagents applied to detect and remove tumour cells from bone marrow harvested for autologous transplantation.

REVERSE TRANSCRIPPASE (RT) ACPIVITY AND RETROVIRAL SEROLOGY IN KAWASAKI DISEASE (KD)

We have recently published evidence that a retrovirus nay be etiologically related to KD (Lancet, Sept. 6, 1986). We have performed co-cultivation of acute KD patient peripheral blood mcnonuclear cells. (PMBC's) with the human lymphoblastoid cell lines MOLT-3, MOLT-4, NC-37, and HUT-78 in RPMI 1640 media containing PHA, IL-2 and in some cases, polybrene, anti-αinterferon, and PHA-stimulated normal adult or cord blood PBMC's. Culture supernatants were assayed weekly for Mg++-dependent RT activity: aliquots were centrifuged, and the pellet resuspended in tris-HC1 with KC1, DTT, MgCl2, Triton X-100, poly(rA) oligo(dT) and 3H-TTP. After incubation, duplicate aliquots were spotted onto glass fiber filters, washed, dried, and counted. RT activity was detected in 16/341 KD co-cultivation flask supernatants but in only 2/278 flasks containing control lymphocytes (p<0.01). None of 4 convalescent KD sera had antibody to gp120 or gp41, envelope glycoproteins of HIV (HTLV-III), by immunoblot analysis. None of 8 convalescent KD sera had IgG antibody to HTLV-I, and none of 7 had IgM antibody to HTLV-I or IgG antibody to HTLV-II by ELISA's utilizing purified whole disrupted virus. The finding of RT activity in co-cultured supematants supports the possibility of a retroviral etiology for KD, but serologic studies thus far do not show cross-reactive antibodies to HTLV-I, HTLV-II, or HIV in patients with KD.

REVERSE TRANSCRIPTASE ACTIVITY (RTA) IN PERIPHERAL BLOOD MONONUCLEAR CELL (PBMC) CULTURES (CXS) FROM PATIENTS (PTS) WITH KAWASAKI DISEASE (KD)

Polymerase activity has been reported in PBMC cxs of pts with KD (Nature 323:814, 1986). To further define its template specificity and persistence after clinical recovery and gammaglobulin (GG) therapy, we studied 89 cxs from 33 KD pts and 33 controls. Of the KD pts 23/33 (70%) showed RTA above the mean + 2 SD for control pts (5.6 picomoles dTMP incorporated). The mean peak RTA for KD PBMC ex supernatants was 8.2 pmoles ± 1.8 vs. 3.7 pmoles ± 0.5 for the controls (p<.05). GG therapy did not prevent expression of RTA which was detected 3-8 weeks post-treatment in cxs from 4/6 KD pts. RTA was detected in PBMC from pts as late as 9 years following acute KD. Co-cultivation of PBMC or cell-free supernatants from PBMC cxs with the HUT-78 cell line yielded RTA > control mean +2 SD (3.1 pmoles) in 14/18 (78%) attempts. Analysis of template/ primer specificity showed no increase in dTMP incorporation when assay conditions were altered to favor DNA pol I or terminal transferase. Duplicate assays to compare polyrA:oligodT vs. polyrC:oligodG showed comparable activity for the KD cxs. We conclude that the polymerase activity associated with the particulate fraction of supernatants from KD PBMC is consistent with RTA and can be detected late after recovery which suggests a retrovirus as the causative agent of KD.