Dopamine pathway loss in nucleus accumbens and ventral tegmental area predicts apathetic behavior in MPTP-lesioned monkeys.

Apathy, primarily defined as a lack of motivation, commonly occurs in people with Parkinson disease (PD). Although dysfunction of basal ganglia pathways may contribute to apathy, the role of dopamine remains largely unknown. We investigated the role of dopaminergic pathways in the manifestation of apathetic behaviors by measuring the effects of the selective dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on monkeys' willingness to attempt goal directed behaviors, distinct from their ability to perform tasks. Fifteen macaques received variable doses of MPTP, had PET scans with [(11)C]-dihydrotetrabenazine (DTBZ), [(11)C]-2ß-3ß-4-fluorophenyltropane (CFT), and [(18)F]-fluorodopa (FD) and performed tasks to assess apathetic behaviors and motor impairment. At 8 weeks post-MPTP, primates were euthanized and stereological cell counts and dopamine measurements were done. Apathy scores were compared to motor scores, in vitro and in vivo dopaminergic measures. Apathy scores increased following MPTP and correlated with DTBZ (r(S) = -0.85), CFT (r(S) = -0.87), and FD (r(S) = -0.85) specific uptake in nucleus accumbens (NAcc,) and dopaminergic cell counts in ventral tegmental area (VTA, r(S) = -0.80). Dopaminergic cell loss in VTA provided significant predictive power for apathy scores after controlling for the influence of cell loss in SN. Additionally, forward step-wise regression analyses indicated that neuropathological changes in the VTA-NAcc pathway predict apathetic behavior better than motor impairment or neuropathological changes in the nigrostriatal network. Our findings suggest that dopaminergic dysfunction within the VTA-NAcc pathway plays a role in the manifestation of apathetic behaviors in MPTP-lesioned primates. Similar changes in people with PD may contribute to apathy.

Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients.

Epstein-Barr virus (EBV) causes potentially lethal immunoblastic lymphoma in up to 25% of children receiving bone marrow transplants from unrelated or HLA-mismatched donors. Because this complication appears to stem from a deficiency of EBV-specific cytotoxic T cells, we assessed the safety and efficacy of donor-derived polyclonal (CD4(+) and CD8(+)) T-cell lines as immunoprophylaxis and treatment for EBV-related lymphoma. Thirty-nine patients considered to be at high risk for EBV-induced lymphoma each received 2 to 4 intravenous infusions of donor-derived EBV-specific T lymphocytes, after they had received T-cell-depleted bone marrow from HLA-matched unrelated donors (n = 33) or mismatched family members (n = 6). The immunologic effects of this therapy were monitored during and after the infusions. Infused cells were identified by detection of the neo marker gene. EBV-specific T cells bearing the neo marker were identified in all but 1 of the patients. Serial analysis of DNA detected the marker gene for as long as 18 weeks in unmanipulated peripheral blood mononuclear cells and for as long as 38 months in regenerated lines of EBV-specific cytotoxic T cells. Six patients (15.5%) had greatly increased amounts of EBV-DNA on study entry (>2, 000 genome copies/10(6) mononuclear cells), indicating uncontrolled EBV replication, a complication that has had a high correlation with subsequent development of overt lymphoma. All of these patients showed 2 to 4 log decreases in viral DNA levels within 2 to 3 weeks after infusion and none developed lymphoma, confirming the antiviral activity of the donor-derived cells. There were no toxic effects that could be attributed to prophylactic T-cell therapy. Two additional patients who did not receive prophylaxis and developed overt immunoblastic lymphoma responded fully to T-cell infusion. Polyclonal donor-derived T-cell lines specific for EBV proteins can thus be used safely to prevent EBV-related immunoblastic lymphoma after allogeneic marrow transplantation and may also be effective in the treatment of established disease.

Adoptive immunotherapy for Epstein-Barr virus-related lymphoma.

Epstein-Barr virus (EBV) causes opportunistic B cell lymphomas in patients whose cellular immunity is compromised. We have been investigating whether infusions of donor-derived, EBV-specific cytotoxic T cells can prevent and/or treat EBV-related lymphoproliferative disease in children receiving T cell-depleted bone marrow from HLA-matched, unrelated or HLA-mismatched, related donors. In this review, we discuss the rationale for this therapeutic approach, describe our experiences with the regimen thus far, and consider some future directions in immunotherapy.

Long-term restoration of immunity against Epstein-Barr virus infection by adoptive transfer of gene-modified virus-specific T lymphocytes.

Adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTLs) offers safe and effective therapy for certain viral infections and could prove useful in the eradication of tumor cells. Whether or not the infused T cells persist for extended periods, retaining their ability to expand in response to antigenic stimulation, is not known. We now report long-term detection of gene-marked Epstein-Barr virus (EBV)-specific CTLs in immunocompromised patients at risk for the development of EBV lymphoproliferative disease. Infusions of CTLs not only restored cellular immune responses against EBV, but also established populations of CTL precursors that could respond to in vivo or ex vivo challenge with the virus for as long as 18 months. Our findings support wider use of antigen-specific CTLs in adoptive immunotherapy.

Production of genetically modified Epstein-Barr virus-specific cytotoxic T cells for adoptive transfer to patients at high risk of EBV-associated lymphoproliferative disease.

EBV-induced lymphoproliferative disease (EBV-LPD) is a disorder most commonly associated with the immunocompromise that follows allogeneic organ transplantation. In patients receiving T cell-depleted bone marrow from HLA-mismatched or HLA-matched unrelated donors, the incidence of EBV-LPD is particularly high, ranging from 5 to 30%. Administration of EBV-specific cytotoxic T lymphocytes may be one means of preventing and treating this disease. We now describe a method that allows the routine and timely preparation of large numbers of such cells to allow their safe administration to bone marrow transplant recipients. We also describe how these cells may be genetically marked before infusion, to determine their fate and disposition in vivo.

Early identification of Epstein-Barr virus-associated post-transplantation lymphoproliferative disease.

Epstein-Barr virus-associated lymphoproliferative disease (EBV-LPD) is a common, usually fatal, complication developing after transplantation of bone marrow from HLA-mismatched or HLA-matched unrelated donors. Prompted by recent reports of successful treatment of EBV-LPD, we investigated methods which could result in early identification of patients at high risk for this disorder, thus improving the likelihood of successful therapeutic interventions. Both the outgrowth of transformed B lymphocytes ex vivo (100% correlation) and the detection of EBV DNA by a PCR method (80% correlation) showed statistically significant association with the histopathological diagnosis of EBV-LPD. Because these abnormalities can be detected prior to the onset of clinical disease. It should now be possible to use a combination of the methods described here to identify patients at high risk of developing EBV-LPD, thus enabling early therapeutic intervention.

Factors that influence the therapeutic activity of 5-fluorouracil [6RS]leucovorin combinations in colon adenocarcinoma xenografts.

The therapeutic activity of FUra alone or combined with [6RS]LV doses ranging from 50 to 1,000 mg/m2 was examined in eight colon adenocarcinoma xenografts, of which five were established from adult neoplasms (HxELC2, HxGC3, HxVRC5, HxHC1, and HxGC3/c1TK-c3 selected for TK deficiency) and three were derived from adolescent tumors (HxSJC3A, HxSJC3B, and HxSJC2). The growth-inhibitory effects of FUra were potentiated by higher doses of [6RS]LV (500-1,000 mg/m2) in three lines (HxGC3/c1TK-c3, HxSJC3A, and HxSJC3B) and by a low dose of [6RS]LV in only one tumor (HxVRC5). Expansion of pools of CH2-H4PteGlun+H4PteGlun (greater than or equal to 2.4-fold) in response to higher doses of [6RS]LV was obtained in all lines except HxHC1. Metabolism of [6RS]LV was high in HxVRC5, with high levels of 5-CH3-H4PteGlu being detected, but not in HxHC1, in which levels of 5-CH3-H4PteGlu and CH = H4PteGlu+10-CHO-H4PteGlu remained relatively low. In the adolescent tumors, levels of CH = H4PteGlu+10-CHO-H4PteGlu were consistently higher than those of 5-CH3-H4PteGlu following [6RS]LV administration, and in HxSJC3A, in which pools of CH2-H4PteGlun+H4PteGlun were significantly expanded, 5-CH3-H4PteGlu concentrations were lower than those observed in the other two lines. The sensitivity of tumors to FUra +/- [6RS]LV and the characteristics of [6S]LV metabolism did not correlate with the activity of CH = H4PteGlu synthetase, the enzyme responsible for the initial cellular metabolism of [6S]LV to CH = H4PteGlu. Thus, no single metabolic phenotype correlated with the [6RS]LV-induced expansion of CH2-H4PteGlun+H4PteGlun pools. Potentiation of the therapeutic efficacy of FUra by [6RS]LV was observed in HxGC3/c1TK-c3 xenografts but not in parent HxGC3 tumors, demonstrating the influence of dThd salvage capability in the response to FUra-[6RS]LV combinations. Plasma dThd concentrations in CBA/CaJ mice were high (1.1 microM). The present data therefore demonstrate the importance of (1) higher doses of [6RS]LV, (2) expansion of pools of CH2-H4PteGlun+H4PteGlun, and (3) dThd salvage capability in potentiation of the therapeutic efficacy of FUra in colon adenocarcinoma xenografts. The plasma levels of FUra achieved in mice are presented.

Accumulation, intracellular metabolism, and antitumor activity of high- and low-dose methotrexate in human osteosarcoma xenografts.

High doses of methotrexate with leucovorin rescue are routinely used in the treatment of patients with osteosarcoma; the rationale for this application is controversial. Using human osteosarcoma xenografts growing in mice as a clinically relevant model, we compared the accumulation, intracellular metabolism, and tumor response of methotrexate administered as either high-dose (2400 mg/kg) or low-dose (150 mg/kg) infusions. The high-dose regimen, which included i.v. hydration and leucovorin rescue, resulted in plasma methotrexate levels that approximated those in patients receiving the drug at 12 g/m2. The low-dose infusion produced essentially the same toxicity as the higher dose level, without use of leucovorin. The HxOs33 tumor line was moderately sensitive to the high-dose infusion (55-day delay in tumor volume doubling time), whereas the second line, HxOs2, did not respond. Neither xenograft had a measurable response to low-dose methotrexate. Methotrexate was present in both tumors for up to 72 hr post-infusion, regardless of the dosage regimen. Only shorter-chain polyglutamates (MTXglu2 and MTXglu3) were detected over this period in the high-dose trial, and levels of these derivatives were uniformly higher in the resistant HxOs2 xenograft. Low-dose infusions were associated with formation of longer-chain polyglutamate species, with more abundant production in the HxOs2 line. Methotrexate polyglutamates exceeded baseline [3H]MTX binding of dihydrofolate reductase, as measured in tumor homogenates, at all testing intervals through 72 hr in both tumor lines. Nonetheless, high-dose methotrexate-induced suppression of [14C]formate incorporation into DNA was greater in the drug-sensitive HxOs33 tumor than in HxOs2. These results suggest a therapeutic advantage for high-dose methotrexate regimens in the treatment of human osteosarcoma but show that formation of tumor MTX polyglutamates is not the sole determinant of response to this agent.

Photolysis of the lysosomal neuraminidase in cultured human skin fibroblasts in the presence of a photoreactive competitive inhibitor.

Photolysis of the lysosomal neuraminidase in crude homogenates of cultured human skin fibroblasts was carried out using the potent competitive enzyme inhibitor, 9-S-(4-azido-2-nitrophenyl)-5-acetamido-2,6-anhydro-2,3,5,9-tetradeoxy-9 -thio-D - glycero-D-galacto-non-2-enonic acid (9-PANP-2,3-D-NANA). Irradiation of the homogenate and the inhibitor (2 min, pH 4.3, 10 degrees C) with a medium pressure mercury lamp resulted in about a 24% reduction of enzyme activity compared to irradiated controls that did not contain additives. No significant loss of activity was observed with homogenate that contained a photoreactive thioglycoside of sialic acid that was not an inhibitor of the enzyme. Similarly, the enzyme activity was not affected when 2-deoxy-2,3-dehydro-N-acetyl neuraminic acid was photolyzed with the homogenate. The latter is a potent competitive inhibitor but it is not photoreactive. Also, the products obtained by prephotolyzing 9-PANP-2,3-D-NANA gave similar enzyme levels under standard assay conditions when compared with the nonirradiated material. Together, these results demonstrate that the photoinactivation is highly specific and both the aryl azide and the unsaturated pyran portion of the molecule are required for inactivation. The title compound may be useful as a potential photolabeling reagent which may facilitate purification of the enzyme and permit further characterization of the mutation in sialidosis patients.