Overall survival by clinical risk category for high dose interleukin-2 (HD IL-2) treated patients with metastatic renal cell cancer (mRCC): data from the PROCLAIMSM registry.

BACKGROUND: Prognostic scoring systems are used to estimate the risk of mortality from metastatic renal cell carcinoma (mRCC). Outcomes from different therapies may vary within each risk group. These survival algorithms have been applied to assess outcomes in patients receiving T-cell checkpoint inhibitory immunotherapy and tyrosine kinase inhibitor therapy, but have not been applied extensively to patients receiving high dose interleukin-2 (HD IL-2) immunotherapy. METHODS: Survival of 810 mRCC patients treated from 2006 to 2017 with high dose IL-2 (aldesleukin) and enrolled in the PROCLAIMSM registry data base was assessed utilizing the International Metastatic RCC Database Consortium (IMDC) risk criteria. Median follow-up is 23.4 months (mo.) (range 0.2-124 mo.). Subgroup evaluations were performed by separating patients by prior or no prior therapy, IL-2 alone, or therapy subsequent to IL-2. Some patients were in two groups. We will focus on the 356 patients who received IL-2 alone, and evaluate outcome by risk factor categories. RESULTS: Among the 810 patients, 721 were treatment-naïve (89%) and 59% were intermediate risk. Overall, of the 249 patients with favorable risk, the median overall survival (OS) is 63.3 mo. and the 2-year OS is 77.6%. Of 480 patients with intermediate risk, median OS is 42.4 mo., 2-year OS 68.2%, and of 81 patients with poor risk, median OS 14 mo., 2-year OS 40.4%. Among those who received IL-2 alone (356 patients), median OS is 64.5, 57.6, and 14 months for favorable, intermediate and poor risk categories respectively. Two year survival among those treated only with HD IL-2 is 73.4, 63.7 and 39.8%, for favorable, intermediate and poor risk categories respectively. CONCLUSIONS: Among mRCC patients treated with HD IL-2, all risk groups have median and 2-year survival consistent with recent reports of checkpoint or targeted therapies for mRCC. Favorable and intermediate risk (by IMDC) patients treated with HD IL-2 have longer OS compared with poor risk patients, with most durable OS observed in favorable risk patients. Favorable risk patients treated with HD IL-2 alone have a 2-year OS of 74%. These data continue to support a recommendation for HD IL-2 for patients with mRCC who meet eligibility criteria. TRIAL REGISTRATION: PROCLAIM, NCT01415167 was registered with ClinicalTrials.gov on August 11, 2011, and initiated for retrospective data collection until 2006, and prospective data collection ongoing since 2011.

Manipulating the host response to autologous tumour vaccines.

From our way of thinking the problem facing vaccine strategies for cancer is not that we do not have "enough" tumour antigens. The problem is we cannot induce an immune response that is sufficient to mediate tumour regression. The normal "checks and balances" found in the body prevent the sustained expansion and subsequent persistence of immune killer cells. If vaccine strategies are going to become effective treatments for cancer patients, they will need to overcome this substantial roadblock. Recent developments in immunology have provided insights into the mechanisms that regulate the expansion and persistence of T cells. This has allowed investigators to reinterpret decades-old observations suggesting that chemotherapy administered before vaccination often led to a stronger immune response. This manuscript will review experiments that offer an explanation for these observations and present pre-clinical data from our laboratory that describes an innovative new approach to combining chemotherapy and vaccination. This approach is readily translatable to the clinic and is broadly applicable to any vaccine strategy for advanced cancer.

Glutamate synthase: a fascinating pathway from L-glutamine to L-glutamate.

Glutamate synthase is a multicomponent iron-sulfur flavoprotein belonging to the class of N-terminal nucleophile amidotransferases. It catalyzes the conversion of L-glutamine and 2-oxoglutarate into two molecules of L-glutamate. In recent years the X-ray structures of the ferredoxin-dependent glutamate synthase and of the a subunit of the NADPH-dependent glutamate synthase have become available. Thanks to X-ray crystallography, it is now known that the ammonia reaction intermediate is transferred via an intramolecular tunnel from the amidotransferase domain to the synthase domain over a distance of about 32A. Although ammonia channeling is a recurrent theme for N-terminal nucleophile and triad-type amidotransferases, the molecular mechanisms of ammonia transfer and its control are different for each known amidotransferase. This review focuses on the intriguing mechanism of action and self-regulation of glutamate synthase with a special focus on the structural data.

Determination of the midpoint potential of the FAD and FMN flavin cofactors and of the 3Fe-4S cluster of glutamate synthase.

Glutamate synthase is a complex iron-sulfur flavoprotein that catalyzes the reductive transfer of the L-glutamine amide group to C(2) of 2-oxoglutarate, forming two molecules of L-glutamate. The bacterial enzyme is an alphabeta protomer, which contains one FAD (on the beta subunit, approximately 50 kDa), one FMN (on the alpha subunit, approximately 150 kDa), and three different Fe-S clusters (one 3Fe-4S center on the alpha subunit and two 4Fe-4S clusters at an unknown location). To address the problem of the intramolecular electron pathway, we have measured the midpoint potential values of the flavin cofactors and of the 3Fe-4S cluster of glutamate synthase in the isolated alpha and beta subunits and in the alphabeta holoenzyme. No detectable amounts of flavin semiquinones were observed during reductive titrations of the enzyme, indicating that the midpoint potential value of each flavin(ox)/flavin(sq) couple is, in all cases, significantly more negative than that of the corresponding flavin(sq)/flavin(hq) couple. Association of the two subunits to form the alphabeta protomer does not alter significantly the midpoint potential value of the FMN cofactor and of the 3Fe-4S cluster (approximately -240 and -270 mV, respectively), but it makes that of FAD some 40 mV less negative (approximately -340 mV for the beta subunit and -300 mV for FAD bound to the holoenzyme). Binding of the nonreducible NADP(+) analogue, 3-aminopyridine adenine dinucleotide phosphate, made the measured midpoint potential value of the FAD cofactor approximately 30-40 mV less negative in the isolated beta subunit, but had no effect on the redox properties of the alphabeta holoenzyme. This result correlates with the formation of a stable charge-transfer complex between the reduced flavin and the oxidized pyridine nucleotide in the isolated beta subunit, but not in the alphabeta holoenzyme. Binding of L-methionine sulfone, a glutamine analogue, had no significant effect on the redox properties of the enzyme cofactors. On the contrary, 2-oxoglutarate made the measured midpoint potential value of the 3Fe-4S cluster approximately 20 mV more negative in the isolated alpha subunit, but up to 100 mV less negative in the alphabeta holoenzyme as compared to the values of the corresponding free enzyme forms. These findings are consistent with electron transfer from the entry site (FAD) to the exit site (FMN) through the 3Fe-4S center of the enzyme and the involvement of at least one of the two low-potential 4Fe-4S centers, which are present in the glutamate synthase holoenzyme, but not in the isolated subunits. Furthermore, the data demonstrate a specific role of 2-oxoglutarate in promoting electron transfer from FAD to the 3Fe-4S cluster of the glutamate synthase holoenzyme. The modulatory role of 2-oxoglutarate is indeed consistent with the recently determined three-dimensional structure of the glutamate synthase alpha subunit, in which several polypeptide stretches are suitably positioned to mediate communication between substrate binding sites and the enzyme redox centers (FMN and the 3Fe-4S cluster) to tightly control and coordinate the individual reaction steps [Binda, C., et al. (2000) Structure 8, 1299-1308].

A prospective randomized phase II trial of GM-CSF priming to prevent topotecan-induced neutropenia in chemotherapy-naive patients with malignant melanoma or renal cell carcinoma.

We conducted a phase II randomized trial of recombinant granculocyte-macrophage colony-stimulating factor (GM-CSF) administered before topotecan chemotherapy to determine whether it could prevent myelosuppression and to determine the antitumor activity of this topoisomerase I inhibitor in 53 patients with metastatic malignant melanoma and renal cell cancer. All patients received GM-CSF after topotecan at a dose of 250 microg/m(2) daily for at least 8 days. Patients randomly assigned to receive GM-CSF priming were treated with GM-CSF at 250 microg/m(2) twice daily for 5 days before treatment. Twenty-five patients were randomly assigned to receive GM-CSF priming and 28 to receive topotecan without priming. The primary analysis was restricted to the protective effects seen during the first cycle of therapy. Grade 4 neutropenia occurred in 8 of 23 patients (35%) and grade 3 neutropenia in 5 of 23 patients (22%) randomized to GM-CSF priming, whereas 18 of 26 (69%) and 5 of 26 (19%) patients experienced grade 4 or 3 neutropenia, respectively, without GM-CSF priming (P =.0074). The mean duration of neutropenia was reduced by GM-CSF priming: grade 3 neutropenia from 5.2 +/- 0.7 to 2.8 +/- 0.7 days (P =.0232) and grade 4 neutropenia from 2.7 +/- 0.6 to 1.1 +/- 0.4 days (P = 0.0332). The protective effects of GM-CSF extended to the second cycle of treatment. The incidence of febrile neutropenia was also reduced. Chemotherapy-induced anemia and thrombocytopenia were similar in both groups. One partial response was seen in a patient with melanoma, and one patient with renal cell cancer had complete regression of pulmonary metastases and was rendered disease-free by nephrectomy. (Blood. 2001;97:1942-1946)

Combination chemotherapy followed by an immunotoxin (anti-B4-blocked ricin) in patients with indolent lymphoma: results of a phase II study.

The purpose of this article was to evaluate the antitumor effects of a combination chemotherapy program based on ProMACE (prednisone, methotrexate, doxorubicin [Adriamycin], cyclophosphamide, etoposide) followed by a B cell-specific immunotoxin in the treatment of patients with advanced-stage indolent histology non-Hodgkin's lymphomas. We performed a prospective phase II clinical trial in a referral-based patient population. After confirmation of diagnosis and staging evaluation, 44 patients (10 small lymphocytic lymphoma, 27 follicular lymphoma, 7 mantle cell lymphoma; 30 without prior therapy, 14 previously treated) received six cycles of ProMACE-CytaBOM (cytarabine, bleomycin, vincristine [Oncovin], mechlorethamine) combination chemotherapy (with etoposide given orally daily for five days) followed by a 7-day continuous infusion of anti-B4-blocked ricin immunotoxin at 30 microg/kg/day given every 14 days for up to six cycles. A complete response was achieved in 25 of 44 patients (57%), 21 from the chemotherapy alone, 3 converted from partial to complete response with the immunotoxin, and 1 patient became a complete responder after a surgical procedure to remove an enlarged spleen that was histologically negative for lymphoma. With a median follow-up of 5 years, 14 of 25 complete responders have relapsed (56%); median remission duration was 2 years, and overall survival was 61%. Forty-two percent of the complete responders have been in continuous remission for more than 4 years. The median number of courses of immunotoxin delivered was two usually because of the development of human anti-ricin antibodies. ProMACE-CytaBOM plus anti-B4-blocked ricin does not produce durable complete remissions in the majority of patients with indolent lymphoma. However, the remissions appear quite durable (> 4 years) in about 40% of the complete responders.

Functional properties of recombinant Azospirillum brasilense glutamate synthase, a complex iron-sulfur flavoprotein.

Azospirillum brasilense glutamate synthase is a complex iron-sulfur flavoprotein that catalyses the NADPH-dependent reductive transfer of glutamine amide group to the C(2) carbon of 2-oxoglutarate to yield L-glutamate. Its catalytically active alphabeta protomer is composed of two dissimilar subunits (alpha subunit, 164.2 kDa; beta subunit, 52.3 kDa) and contains one FAD (at Site 1, the pyridine nucleotide site within the beta subunit), one FMN (at Site 2, the 2-oxoglutarate/L-glutamate site in the alpha subunit) and three different iron-sulfur clusters (one 3Fe-4S center on the alpha subunit and two 4Fe-4S clusters of unknown location). A plasmid harboring the gltD and gltB genes, the genes encoding the glutamate synthase beta and alpha subunits, respectively, each one under the control of the T7/lac promoter of pET11a was found to be suitable for the overproduction of glutamate synthase holoenzyme in Escherichia coli BL21(DE3) cells. Recombinant A. brasilense glutamate synthase could be purified to homogeneity from overproducing E. coli cells by ion exchange chromatography, gel filtration and affinity chromatography on a 2',5' ADP-Sepharose 4B column. The purified enzyme was indistinguishable from that prepared from Azospirillum cells with respect to cofactor content, N-terminal sequence of the subunits, aggregation state, kinetic and spectroscopic properties. The study of the recombinant holoenzyme allowed us to establish that the tendency of glutamate synthase to form a stable (alphabeta)4 tetramer at high protein concentrations is a property unique to the holoenzyme, as the isolated beta subunit does not oligomerize, while the isolated glutamate synthase alpha subunit only forms dimers at high protein concentrations. Furthermore, the steady-state kinetic analysis of the glutamate synthase reaction was extended to the study of the effect of adenosine-containing nucleotides. Compounds such as cAMP, AMP, ADP and ATP have no effect on the enzyme activity, while the 2'-phosphorylated analogs of AMP and NADP(H) analogs act as inhibitors of the reaction, competitive with NADPH. Thus, it can be ruled out that glutamate synthase reaction is subjected to allosteric modulation by adenosine containing (di)nucleotides, which may bind to the putative ADP-binding site at the C-terminus of the alpha subunit. At the same time, the strict requirement of a 2'-phosphate group in the pyridine nucleotide for binding to glutamate synthase (GltS) was established. Finally, by comparing the inhibition constants exhibited by a series of NADP+ analogs, the contribution to the binding energy of the various parts of the pyridine nucleotide has been determined along with the effect of substituents on the 3 position of the pyridine ring. With the exception of thio-NADP+, which binds the tightest to GltS, it appears that the size of the substituent is the factor that affects the most the interaction between the NADP(H) analog and the enzyme.

Elevations in serum soluble interleukin-2 receptor levels predict relapse in patients with hairy cell leukemia.

PURPOSE: Interferon-alfa, 2'-deoxycoformycin, and 2-chlorodeoxy-adenosine (2-CdA) are effective in the management of patients with hairy cell leukemia. These agents produce remissions in most patients, but relapses occur with all three drugs. The optimal means to follow patients for relapse after treatment has not been determined. METHODS: We retrospectively examined serial serum soluble interleukin-2 receptor levels (sIL-2R) and absolute granulocyte counts in eight patients with relapsed hairy cell leukemia. All were treated with 2-CdA at the time of relapse. Serum samples were available at 3- to 6-month intervals from 5 to 9 years before relapse and 2-CdA treatment RESULTS: sIL-2R levels increase only in patients who go on to relapse. sIL-2R levels doubled a mean of 17.1 months (range, 4-36 months) before absolute granulocyte count decreased by 50%. DISCUSSION: Demonstration of a rising serum sIL-2R level in patients with hairy cell leukemia identified those with an increased risk of relapse who need more frequent observation than patients who maintain a stable sIL-2R level. Early intervention may ameliorate the toxicity of salvage therapy because disease-related neutropenia may be anticipated.

Glutamate synthase: identification of the NADPH-binding site by site-directed mutagenesis.

To contribute to the understanding of glutamate synthase and of beta subunit-like proteins, which have been detected by sequence analyses, we identified the NADPH-binding site out of the two potential ADP-binding regions found in the beta subunit. The substitution of an alanyl residue for G298 of the beta subunit of Azospirillum brasilense glutamate synthase (the second glycine in the GXGXXA fingerprint of the postulated NADPH-binding site) yielded a protein species in which the flavin environment and properties are unaltered. On the contrary, the binding of the pyridine nucleotide substrate is significantly perturbed demonstrating that the C-terminal potential ADP-binding fold of the beta subunit is indeed the NADPH-binding site of the enzyme. The major effect of the G298A substitution in the GltS beta subunit consists of an approximately 10-fold decrease of the affinity of the enzyme for pyridine nucleotides with little or no effect on the rate of the enzyme reduction by NADPH. By combining kinetic measurements and absorbance-monitored equilibrium titrations of the G298A-beta subunit mutant, we conclude that also the positioning of its nicotinamide portion into the active site is altered thus preventing the formation of a stable charge-transfer complex between reduced FAD and NADP(+). During the course of this work, the Azospirillum DNA regions flanking the gltD and gltB genes, the genes encoding the GltS beta and alpha subunits, respectively, were sequenced and analyzed. Although the Azospirillum GltS is similar to the enzyme of other bacteria, it appears that the corresponding genes differ with respect to their arrangement in the chromosome and to the composition of the glt operon: no genes corresponding to E. coli and Klebsiella aerogenes gltF or to Bacillus subtilis gltC, encoding regulatory proteins, are found in the DNA regions adjacent to that containing gltD and gltB genes in Azospirillum. Further studies are needed to determine if these findings also imply differences in the regulation of the glt genes expression in Azospirillum (a nitrogen-fixing bacterium) with respect to enteric bacteria.

Cross-talk and ammonia channeling between active centers in the unexpected domain arrangement of glutamate synthase.

INTRODUCTION: The complex iron-sulfur flavoprotein glutamate synthase catalyses the reductive synthesis of L-glutamate from 2-oxoglutarate and L-glutamine, a reaction in the plant and bacterial pathway for ammonia assimilation. The enzyme functions through three distinct active centers carrying out L-glutamine hydrolysis, conversion of 2-oxoglutarate into L-glutamate, and electron uptake from an electron donor. RESULTS: The 3.0 A crystal structure of the dimeric 324 kDa core protein of a bacterial glutamate synthase was solved by the MAD method, using the very weak anomalous signal of the two 3Fe-4S clusters present in the asymmetric unit. The 1,472 amino acids of the monomer fold into a four-domain architecture. The two catalytic domains have canonical Ntn-amidotransferase and FMN binding (beta/alpha)8 barrel folds, respectively. The other two domains have an unusual "cut (beta/alpha)8 barrel" topology and an unexpected novel beta-helix structure. Channeling of the ammonia intermediate is brought about by an internal tunnel of 31 A length, which runs from the site of L-glutamine hydrolysis to the site of L-glutamate synthesis. CONCLUSIONS: The outstanding property of glutamate synthase is the ability to coordinate the activity of its various functional sites to avoid wasteful consumption of L-glutamine. The structure reveals two polypeptide segments that connect the catalytic centers and embed the ammonia tunnel, thus being ideally suited to function in interdomain signaling. Depending on the enzyme redox and ligation states, these signal-transducing elements may affect the active site geometry and control ammonia diffusion through a gating mechanism.

Treatment with tumor necrosis factor-alpha and granulocyte-macrophage colony-stimulating factor increases epidermal Langerhans' cell numbers in cancer patients.

Dendritic cells (DCs) initiate primary and stimulate secondary T-cell responses. We conducted a phase I trial of tumor necrosis factor (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in patients with cancer to increase DCs in peripheral blood or skin based on in vitro data that showed that CD34(+) hematopoietic precursors require these cytokines to mature into functional antigen-presenting DCs. Eleven patients were treated for 7 days with GM-CSF, 125 microg/m(2) twice daily as subcutaneous injections, and TNF-alpha as a continuous infusion at dose levels of 25, 50, or 100 microg/m(2)/day. The maximum tolerated dose of TNF-alpha was 50 microg/m(2)/day with this dose of GM-CSF; dose-limiting toxicities occurred in both patients treated with 100 microg/m(2)/day. One became thrombocytopenic and the other had transient confusion. Epidermal Langerhans' cells were quantitated by S100 staining of skin biopsies and DC precursors in peripheral blood by colony-forming unit dendritic (CFU-dendritic) assays. S100-positive cells in the epidermis doubled after treatment (2.55 S100(+) cells/high-power field before treatment to 6.05 after treatment, p = 0.029). CFU-dendritic in peripheral blood increased after treatment in 3 colorectal cancer patients but not in 3 patients with melanoma. CD11c(+) or CD123(+), HLA-DR(bright), lineage-negative dendritic cell precursors were not increased in peripheral blood mononuclear cells. This trial demonstrates that treatment with TNF-alpha and GM-CSF can increase the number of DCs in the skin and the number of dendritic cell precursors in the blood of some patients with cancer. This approach may increase the efficacy of vaccination to tumor antigens in cancer patients.

Glutamate synthase: a complex iron-sulfur flavoprotein.

Glutamate synthase is a complex iron-sulfur flavoprotein that forms L-glutamate from L-glutamine and 2-oxoglutarate. It participates with glutamine synthetase in ammonia assimilation processes. The known structural and biochemical properties of glutamate synthase from Azospirillum brasilense, a nitrogen-fixing bacterium, will be discussed in comparison to those of the ferredoxin-dependent enzyme from photosynthetic tissues and of the eukaryotic reduced pyridine nucleotide-dependent form of glutamate synthase in order to gain insight into the mechanism of the glutamate synthase reaction. Sequence analyses also revealed that the small subunit of bacterial glutamate synthase may be the prototype of a novel class of flavin adenine dinucleotide- and iron-sulfur-containing oxidoreductase widely used as an enzyme subunit or domain to transfer reducing equivalents from NAD(P)H to an acceptor protein or protein domain.

Abrogation of the hematological and biological activities of the interleukin-3/granulocyte-macrophage colony-stimulating factor fusion protein PIXY321 by neutralizing anti-PIXY321 antibodies in cancer patients receiving high-dose carboplatin.

This dose-escalation study was performed to evaluate the hematologic activity, biological effects, immunogenicity, and toxicity of PIXY321 (an interleukin-3/granulocyte-macrophage colony-stimulating factor fusion protein) administered after high-dose carboplatin (CBDCA) treatment. Patients with advanced cancers received CBDCA at 800 mg/m2 intravenously on day 0 of repeated 28-day cycles. In part A of the study, patients were treated with CBDCA alone during cycle 1 and then received PIXY321 on days 1 through 18 of cycle 2 and later cycles. In part B, patients received 18 days of PIXY321 beginning on day 1 of all CBDCA cycles, including cycle 1. PIXY321 was administered subcutaneously in 2 divided doses. Total doses of 135, 250, 500, 750, and 1,000 micrograms/m2/d were administered to successive cohorts of 3 to 6 patients in part A. In part B, patient groups received PIXY321 doses of 750, 1,000, and 1,250 micrograms/m2/d. The hematologic effects of PIXY321 were assessed in the first 2 cycles of therapy. Anti-PIXY321 antibody formation was assessed by enzyme-linked immunosorbent assay (ELISA) and neutralization assay. Of the 49 patients enrolled, 31 were fully evaluable for hematologic efficacy. When comparing the first B cycle (cycle B-1; with PIXY321) with the first A cycle (cycle A-1; without PIXY321), the fusion protein had no significant effect on platelet nadirs or duration of platelets less than 20,000/microL but was able to speed the time of recovery of platelet counts to 100,000/microL (15 v 20 days; P =.01). Significant improvements in neutrophil nadir and duration of ANC less than 500 were observed in cycles A-2 and B-1 (with PIXY321) as compared with cycle A-1 (without PIXY321). Initial PIXY321 prophylaxis (cycle A-2 and cycle B-1), enhanced the recovery of ANC to greater than 1,500/microL by an average of at least 8 days as compared with cycle A-1 (without PIXY321; P

Porcine recombinant dihydropyrimidine dehydrogenase: comparison of the spectroscopic and catalytic properties of the wild-type and C671A mutant enzymes.

Dihydropyrimidine dehydrogenase catalyzes, in the rate-limiting step of the pyrimidine degradation pathway, the NADPH-dependent reduction of uracil and thymine to dihydrouracil and dihydrothymine, respectively. The porcine enzyme is a homodimeric iron-sulfur flavoprotein (2 x 111 kDa). C671, the residue postulated to be in the uracil binding site and to act as the catalytically essential acidic residue of the enzyme oxidative half-reaction, was replaced by an alanyl residue. The mutant enzyme was overproduced in Escherichia coli DH5alpha cells, purified to homogeneity, and characterized in comparison with the wild-type species. An extinction coefficient of 74 mM-1 cm-1 was determined at 450 nm for the wild-type and mutant enzymes. Chemical analyses of the flavin, iron, and acid-labile sulfur content of the enzyme subunits revealed similar stoichiometries for wild-type and C671A dihydropyrimidine dehydrogenases. One FAD and one FMN per enzyme subunit were found. Approximately 16 iron atoms and 16 acid-labile sulfur atoms were found per wild-type and mutant enzyme subunit. The C671A dihydropyrimidine dehydrogenase mutant exhibited approximately 1% of the activity of the wild-type enzyme, thus preventing its steady-state kinetic analysis. Therefore, the ability of the C671A mutant and, for comparison, of the wild-type enzyme species to interact with reaction substrates, products, or their analogues were studied by absorption spectroscopy. Both enzyme forms did not react with sulfite. The wild-type and mutant enzymes were very similar to each other with respect to the spectral changes induced by binding of the reaction product NADP+ or of its nonreducible analogue 3-aminopyridine dinucleotide phosphate. Uracil also induced qualitatively and quantitatively similar absorbance changes in the visible region of the absorbance spectrum of the two enzyme forms. However, the calculated Kd of the enzyme-uracil complex was significantly higher for the C671A mutant (9.1 +/- 0.7 microM) than for the wild-type dihydropyrimidine dehydrogenase (0.7 +/- 0.09 microM). In line with these observations, the two enzyme forms behaved in a similar way when titrated anaerobically with a NADPH solution. Addition of an up to 10-fold excess of NADPH to both dihydropyrimidine dehydrogenase forms led to absorbance changes consistent with reduction of approximately 0.5 flavin per subunit, with no indication of reduction of the enzyme iron-sulfur clusters. Absorbance changes consistent with reduction of both enzyme flavins were obtained by removing NADP+ with a NADPH-regenerating system. On the contrary, the two enzyme species differed significantly with respect to their reactivity with dihydrouracil. Addition of dihydrouracil to the wild-type enzyme species, under anaerobic conditions, led to absorbance changes that could be interpreted to result from both partial flavin reduction and the formation of a complex between the enzyme and (dihydro)uracil. In contrast, only spectral changes consistent with formation of a complex between the oxidized enzyme and dihydrouracil were observed when a C671A mutant enzyme solution was titrated with this compound. Furthermore, enzyme-monitored turnover experiments were carried out anaerobically in the presence of a limiting amount of NADPH and excess uracil with the two enzyme forms in a stopped-flow apparatus. These experiments directly demonstrated that the substitution of an alanyl residue for C671 in dihydropyrimidine dehydrogenase specifically prevents enzyme-catalyzed reduction of uracil. Finally, sequence analysis of dihydropyrimidine dehydrogenase revealed that it exhibits a modular structure; the N-terminal region, similar to the beta subunit of bacterial glutamate synthases, is proposed to be responsible for NADPH binding and oxidation with reduction of the FAD cofactor of dihydropyrimidine dehydrogenase. The central region, similar to the FMN subunit of dihydroorotate dehydrogenases, is likely to harbor the site o

Phase I trial of anti-CD3-stimulated CD4+ T cells, infusional interleukin-2, and cyclophosphamide in patients with advanced cancer.

PURPOSE: We performed a phase I trial to determine whether in vivo expansion of activated CD4+ T cells was possible in cancer patients. 111Indium labeling was used to observe trafficking patterns of the infused stimulated CD4+ T cells. The influence of cyclophosphamide (CTX) dosing on immunologic outcome was also examined. PATIENTS AND METHODS: Patients with advanced solid tumors or non-Hodgkin's lymphoma received CTX at 300 or 1,000 mg/m2 intravenously (i.v.). Leukapheresis was performed to harvest peripheral-blood mononuclear cells (PBMCs) either just before the CTX dose, or when the patient was either entering or recovering from the leukocyte nadir induced by CTX. An enriched population of CD4+ T cells was obtained by negative selection. The CD4+ T cells were activated ex vivo with anti-CD3, cultured with interleukin-2 (IL-2) for 4 days, and adoptively transferred. After adoptive transfer, patients received IL-2 (9.0 x 10(6) IU/m2/d) by continuous infusion for 7 days. RESULTS: The absolute number of CD4+, CD4+/DR+, and CD4+/CD45RO+ T cells increased in a statistically significant fashion in all cohorts after the first course of therapy. The degree of CD4 expansion was much greater than CD8 expansion, which resulted in a CD4:CD8 ratio that increased in 26 of 31 patients. The greatest in vivo CD4 expansion occurred when cells were harvested as patients entered the CTX-induced nadir. One complete response (CR), two partial responses (PRs), and eight minor responses were observed. Trafficking of 111Indium-labeled CD4 cells to subcutaneous melanoma deposits was also documented. CONCLUSION: CD4+ T cells can be expanded in vivo in cancer patients, which results in increased CD4:CD8 ratios. The timing of pheresis in relation to CTX administration influences the degree of CD4 expansion. Tumor responses with this regimen were observed in a variety of tumors, including melanoma and non-Hodgkin's lymphoma; a high percentage of patients had at least some tumor regression from the regimen that produced the greatest CD4+ T-cell expansion.

A phase II study of carboplatin, cisplatin, interferon-alpha, and tamoxifen for patients with metastatic melanoma.

The purpose of this trial was to determine the toxicity and antineoplastic activity of cisplatin, carboplatin, tamoxifen, and interferon-alpha (IFN-alpha) in patients with advanced melanoma. Eleven patients with metastatic melanoma were enrolled. The patients received carboplatin 400 mg/m2 i.v. on day 0; cisplatin 25 mg/m2 i.v. on days 7, 14, and 21; tamoxifen 20 mg p.o. b.i.d. on days 0-27; and interferon-alpha 5 million units/m2 subcutaneously 3 times per week. Cycles were repeated every 28 days. Patients were assessed for tumor response at the end of 2 cycles. Toxicity was severe, with 14 of 24 cycles given requiring some form of dose reduction. Carboplatin dose reductions were related to bone-marrow toxicity, whereas IFN-alpha caused fatigue, arthralgias, myalgias, and fever. The overall response rate was 18% (2 partial responses [PRs]). The combination of cisplatin, carboplatin, tamoxifen, and IFN-alpha is active in advanced melanoma; however, the toxicity is unacceptable.

The recombinant alpha subunit of glutamate synthase: spectroscopic and catalytic properties.

As part of our studies of Azospirillum brasilense glutamate synthase, a complex iron-sulfur flavoprotein, we have overproduced the two enzyme subunits separately in Escherichia coli. The beta subunit (53.2 kDa) was demonstrated to contain the site of NADPH oxidation of glutamate synthase and the FAD cofactor, which was identified as Flavin 1 of glutamate synthase, the flavin located at the site of NADPH oxidation. We now report the overproduction of the glutamate synthase alpha subunit (162 kDa), which is purified to homogeneity in a stable form. This subunit contains FMN as the flavin cofactor which exhibits the properties of Flavin 2 of glutamate synthase: reactivity with sulfite to yield a flavin-N(5)-sulfite addition product (Kd = 2.6 +/- 0.22 mM), lack of reactivity with NADPH, reduction by L-glutamate, and reoxidation by 2-oxoglutarate and glutamine. Thus, FMN is the flavin located at the site of reduction of the iminoglutarate formed on the addition of glutamine amide group to the C(2) carbon of 2-oxoglutarate. The glutamate synthase alpha subunit contains the [3Fe-4S] cluster of glutamate synthase, as shown by low-temperature EPR spectroscopy experiments. The glutamate synthase alpha subunit catalyzes the synthesis of glutamate from L-glutamine and 2-oxoglutarate, provided that a reducing system (dithionite and methyl viologen) is present. The FMN moiety but not the [3Fe-4S] cluster of the subunit appears to participate in this reaction. Furthermore, the isolated alpha subunit of glutamate synthase exhibits a glutaminase activity, which is absent in the glutamate synthase holoenzyme. These findings support a model for glutamate synthase according to which the enzymes prepared from various sources share a common glutamate synthase function (the alpha subunit of the bacterial enzyme, or its homologous polypeptide forming the ferredoxin-dependent plant enzyme) but differ for the chosen electron donor. The pyridine nucleotide-dependent forms of the enzyme have recruited a FAD-dependent oxidoreductase (the bacterial beta subunit) to mediate electron transfer from the NAD(P)H substrate to the glutamate synthase polypeptide. However, it appears that the presence of the enzyme beta subunit and/or of the additional iron-sulfur clusters (Centers II and III) of the bacterial glutamate synthase is required for communication between Center I (the [3Fe-4S] center) and the FMN moiety within the alpha subunit, and for ensuring coupling of glutamine hydrolysis to the transfer of the released ammonia molecule to 2-oxoglutarate in the holoenzyme.

Development of rheumatoid arthritis after treatment of large granular lymphocyte leukemia with deoxycoformycin.

The association of T-cell large granular lymphocyte (LGL) leukemia and rheumatoid arthritis is well described and it is now recognized that these patients and patients with Felty's syndrome represent different aspects of a single disease process. Most patients have rheumatoid arthritis at the time of diagnosis of LGL leukemia. This is the first detailed report of the development of rheumatoid arthritis after the diagnosis and treatment of LGL leukemia as well as the first report of rheumatoid arthritis that occurred in association with deoxycoformycin treatment. It is likely that the beneficial sustained normalization of neutrophil counts as a result of deoxycoformycin treatment played a significant role in the development of this complication. Hematological improvement occurred despite molecular genetic evidence of persistence of the abnormal T-cell clone. The role of the clonally expanded T cells in the pathogenesis of neutropenia and rheumatoid arthritis is discussed.