Efficacy and safety of adalimumab as monotherapy in patients with rheumatoid arthritis for whom previous disease modifying antirheumatic drug treatment has failed.

OBJECTIVE: To evaluate the efficacy and safety of monotherapy with adalimumab in patients with RA for whom previous DMARD treatment has failed. METHODS: In a 26 week, double blind, placebo controlled, phase III trial, 544 patients with RA were randomised to monotherapy with adalimumab 20 mg every other week, 20 mg weekly, 40 mg every other week, 40 mg weekly, or placebo. The primary efficacy end point was > or =20% improvement in the ACR core criteria (ACR20 response). Secondary efficacy end points included ACR50, ACR70, EULAR responses, and the Disability Index of the Health Assessment Questionnaire (HAQ DI). RESULTS: After 26 weeks, patients treated with adalimumab 20 mg every other week, 20 mg weekly, 40 mg every other week, and 40 mg weekly had significantly better response rates than those treated with placebo: ACR20 (35.8%, 39.3%, 46.0%, 53.4%, respectively v 19.1%; p< or =0.01); ACR50 (18.9%, 20.5%, 22.1%, 35.0% v 8.2%; p< or =0.05); ACR70 (8.5%, 9.8%, 12.4%, 18.4% v 1.8%; p< or =0.05). Moderate EULAR response rates were significantly greater with adalimumab than with placebo (41.5%, 48.2%, 55.8%, 63.1% v 26.4%; p< or =0.05). Patients treated with adalimumab achieved better improvements in mean HAQ DI than those receiving placebo (-0.29, -0.39, -0.38, -0.49 v -0.07; p< or =0.01). No significant differences were found between adalimumab and placebo treated patients for serious adverse events, serious infections, or malignancies. Injection site reaction occurred in 10.6% and 0.9% of adalimumab and placebo treated patients, respectively (p< or =0.05). CONCLUSION: Among patients with RA for whom previous DMARD treatment had failed, adalimumab monotherapy achieved significant, rapid, and sustained improvements in disease activity and improved physical function and was safe and well tolerated.

A multicenter, open-label, prospective, randomized, dose-ranging pharmacokinetic study of the anti-TNF-alpha antibody afelimomab in patients with sepsis syndrome.

OBJECTIVE: To investigate the pharmacokinetics and safety of afelimomab, a murine antibody fragment against human tumor necrosis factor (TNF)-alpha in patients with sepsis. DESIGN: Multicenter, randomized, open-label, placebo-controlled phase I/II clinical trial. SETTING: Intensive care units of six academic medical centers in the United States. PATIENTS: Forty-eight patients with a clinical diagnosis of sepsis who received standard supportive care and antimicrobial therapy. INTERVENTIONS: Patients received 0.3, 1.0, or 3.0 mg/kg afelimomab or placebo intravenously over 20 min. Three patients in each dose group received single doses; the remaining nine patients in each group received multiple (nine) doses at 8-h intervals over 72 h. MEASUREMENTS AND MAIN RESULTS: Afelimomab appeared safe and well tolerated. Single- and multiple-dose kinetics were predictable and dose related. The elimination half-life was 44.7 h. Afelimomab treatment resulted in increased serum concentrations of TNF (includes TNF-antibody complexes) and decreased serum interleukin-6 concentrations, whereas no discernible trends were observed in placebo-treated patients. There was no significant treatment effect on 28-day mortality as was expected given the small number of patients. However, overall mortality was significantly (p = 0.001) associated with baseline interleukin-6 concentration. All patients experienced adverse events, but the vast majority were considered unrelated to the study drug and demonstrated no apparent relationship to afelimomab dose. Although 41% of patients developed human anti-murine antibodies, there were no clinical sequelae. CONCLUSIONS: Multidose therapy with afelimomab was safe, well tolerated, and had predictable linear kinetics. A large randomized trial comparing afelimomab to placebo in patients with well defined sepsis has recently been completed.

Phase I and pharmacokinetic study of the water-soluble dolastatin 15 analog LU103793 in patients with advanced solid malignancies.

PURPOSE: To determine the maximum-tolerated dose (MTD), dose-limiting toxicities (DLTs), and pharmacokinetic profile of the dolastatin 15 analog LU103793 when administered daily for 5 days every 3 weeks. PATIENTS AND METHODS: Fifty-six courses of LU103793 at doses of 0.5 to 3.0 mg/m2 were administered to 26 patients with advanced solid malignancies. Pharmacokinetic studies were performed on days 1 and 5 of course one. Pharmacokinetic variables were related to the principal toxicities. RESULTS: Neutropenia, peripheral edema, and liver function test abnormalities were dose-limiting at doses greater than 2.5 mg/m2 per day. Four of six patients developed DLT at 3.0 mg/m2 per day, whereas two of 12 patients treated at 2.5 mg/m2 per day developed DLT. Pharmacokinetic parameters were independent of dose and similar on days 1 and 5. Volume of distribution at steady-state (Vss) was 7.6 +/- 2.0 L/m2, clearance 0.49 +/- 0.18 L/h/m2, and elimination half-life (t1/2) 12.3 +/- 3.8 hours. Peak concentrations (Cmax) on day 1 related to mean percentage decrement in neutrophils (sigmoid maximum effect (Emax) model). Patients who experienced dose-limiting neutropenia had significantly higher Cmax values than patients who did not, whereas nonhematologic DLTs were more related to dose. CONCLUSION: The recommended dose for phase II evaluations of LU103793 daily for 5 days every 3 weeks is 2.5 mg/m2 per day. The lack of prohibitive cardiovascular effects and the generally acceptable toxicity profile support the rationale for performing disease-directed evaluations of LU103793 on the schedule evaluated in this study.

Leukocyte chemotactic activity of cyclophilin.

During the purification of eosinophil chemotactic factors synthesized by the uterus in response to estrogen we isolated a protein having an N-terminal (15 amino acids) sequence identical to that of rat cyclophilin. Our data demonstrate that cyclophilin, a cytosolic protein isolated from bovine thymocytes, which specifically binds the immunosuppressive drug cyclosporin A, as well as recombinant human cyclophilin, displays eosinophil chemotactic activity. In addition to its chemotactic activity, cyclophilin stimulated the release of peroxidase activity from eosinophils. Maximal chemotactic activity of cyclophilin was achieved at a concentration of approximately 10 nM. At similar concentrations cyclophilin was also able to stimulate the migration of neutrophils. This chemotactic activity could be prevented by the addition of cyclosporin A, but not by a nonimmunosuppressive analog (1-fur-furyl-cyclosporin A) at similar concentrations. This chemotactic activity may represent an additional mechanism by which immunosuppressive drugs function to prevent tissue rejection.

Eosinophil differentiation of human umbilical cord mononuclear cells and prolonged survival of mature eosinophils by murine EL-4 thymoma cell conditioned medium.

Umbilical cord mononuclear cells, HL-60 cells, HL-60 clones selected for eosinophil differentiation, and the eosinophil leukemia cell line EoL were tested for their ability to produce eosinophil peroxidase. HL-60 clones selected for eosinophil differentiation produced eosinophil peroxidase, as judged by staining of cells for cyanide-resistant peroxidase activity; however, these cells lost their ability to produce eosinophil peroxidase in long-term culture. In contrast, eosinophil precursors from human umbilical cord blood mononuclear cells stimulated with murine EL-4 conditioned medium (EL-4 CM) were regularly induced to eosinophil protein synthesis, including eosinophil peroxidase, major basic protein, eosinophil cationic protein, and eosinophil-derived neurotoxin, as assessed by cyanide-resistant peroxidase and immunofluorescence staining. This induction by EL-4 CM is either at the level of gene transcription or mRNA stabilization, as shown by the increase of total mRNA for eosinophil peroxidase, major basic protein, and eosinophil-derived neurotoxin by Northern blot analyses. Purified peripheral blood eosinophils incubated for 4 days with EL-4 CM had increased survival over control eosinophils. Moreover, this enhanced survival was specifically blocked by antiserum to interleukin 5. Our results suggest that the effects of EL-4 CM on human umbilical cord mononuclear cells and mature eosinophils are due to the presence of interleukin 5.

Characterization of interleukin 5 receptors on eosinophilic sublines from human promyelocytic leukemia (HL-60) cells.

The T cell product interleukin 5 (IL-5) has been shown to be a key factor in the development and the maturation of the eosinophilic cell lineage. We report here on the detection of human IL-5 receptors on eosinophilic sublines of the promyelocytic leukemia HL-60. Sodium butyrate, which initiates differentiation to mature eosinophils, also induces the appearance of high affinity (Kd 1-5 X 10(-11) M) IL-5 binding sites on these cells. The receptors are specific for IL-5, since binding of radiolabeled ligand can only be inhibited with homologous or murine IL-5 and not by other cytokines. We further show that the receptors are functional, since IL-5 can stimulate the proliferation of these cells. Affinity crosslinking of surface-bound 125I human IL-5 or 35S mouse IL-5 identified two membrane polypeptides of approximately 60 and approximately 130 kD to which IL-5 is closely associated. The presence of granulocyte/macrophage-colony-stimulating factor or tumor necrosis factor during butyrate induction decreased the expression of IL-5 binding sites compared with control cultures. The identification and characterization of human IL-5 receptors on HL-60 sublines should provide new insight into the role of this cytokine in eosinophil differentiation.

Glucocorticoid and progestin regulation of eosinophil chemotactic factor and complement C3 in the estrogen-treated rat uterus.

To study the steroid regulation of estradiol-induced uterine eosinophil chemotactic factor activity (ECF-U) in the rat, we injected immature female rats with combinations of estradiol, dexamethasone, progesterone, and the antihormones RU-486 and ketoconazole. An in vitro chemotactic system using blind well chambers and employing eosinophil-differentiated HL-60 promyelocytic cells was used as a bioassay for ECF-U activity. Dexamethasone and progesterone both antagonized the effects of estradiol on stimulation of ECF-U activity; neither hormone induced ECF-U activity when given alone. RU-486 and ketoconazole were able to block the inhibitory effects of dexamethasone and progesterone. The effects of dexamethasone and progesterone appear to be mediated through their specific nuclear receptors and are not due to direct effects on the chemotactic cell. Comparison of the steroid regulation of ECF-U activity with the estradiol-induced synthesis and secretion of complement component C3 showed that progesterone antagonized the effects of estradiol in both systems, whereas dexamethasone was antagonistic on ECF-U, but not on the secretion of C3. Taken together these results suggest that estradiol's effects in the rat uterus may be modulated by other steroids in at least two systems, bringing into question the common practice of studying uterine steroid actions by evaluation of a single protein or mRNA.

Chemotactic capabilities of HL-60 human myeloid leukemia cells differentiated to eosinophils.

HL-60 human myeloid leukemia cells can be induced to differentiate into cells with many of the morphological, biochemical, and functional features of mature neutrophils, monocyte/macrophages, and eosinophils. HL-60 neutrophils are known to respond to several chemotactic compounds, but similar data do not exist for HL-60 eosinophils. We studied the chemotactic capabilities of eosinophil-differentiated HL-60 cells using a blind-well chamber technique. HL-60 eosinophils responded to the specific eosinophilotactic tetrapeptides Ala-Gly-Ser-Glu, Val-Gly-Asp-Glu, and Val-Gly-Ser-Glu; to the formylated tripeptide N-formyl-L-Met-L-Leu-L-Phe (fMLP); and to extracts estrogen-treated rat uteri, which contain an eosinophilotactin. Concentration optima for chemotaxis of HL-60 eosinophils were similar to those for normal human and rodent eosinophils. Neutrophil-differentiated HL-60 cells responded only to fMLP, whereas undifferentiated HL-60s showed no chemotactic ability. The time course of development of chemotactic competence paralleled other developmental changes in HL-60 eosinophilic differentiation, suggesting a common control mechanism. We conclude that the ability to respond to specific eosinophilotactic compounds is a feature of HL-60 cells that have differentiated to eosinophils and that this cell line may be used as a model system in studies of eosinophil chemotaxis.

Characterization of sublines of HL-60 human leukemia cells resistant to induction of differentiation by butyric acid.

HL-60 human leukemia cells undergo terminal differentiation when cultured with butyric acid. To produce cells resistant to the maturation-inducing effects of butyric acid, two strategies were followed. First, HL-60 cells were mutagenized and cultured in soft agar with inducing concentrations of butyric acid. Four clones were isolated resistant to a wide variety of differentiation inducers, including butyric acid. Second, HL-60 cells were cultured in gradually increasing concentrations of butyric acid until a normal growth rate was achieved in medium containing greater than 1 mM butyric acid. These cells retained their ability to be induced to mature to neutrophils with dimethylsulfoxide, retinoic acid, and actinomycin D; and to monocyte/macrophage like cells with tetradecanoylphorbol acetate and 1,25-(OH)2 vitamin D3. However, they no longer underwent terminal differentiation when butyric acid was added in increasing concentration, even when cytotoxic concentrations were used. The mutagenized clones cells appeared permanently butyrate resistant, but the selected clones reverted to the wild-type state when grown in the absence of butyric acid. The selected cells continued to express myeloperoxidase; the mutagenized lines did not. Thus, by using two different protocols, HL-60 cells resistant to the cytotoxic and maturation-inducing effects of butyric acid can be produced that have varied phenotypic characteristics.

Lineage directed HL-60 cell sublines as a model system for the study of early events in lineage determination of myeloid cells.

Current experimental models are poorly suited to study the early biochemical and molecular events of the lineage determination process in myeloid progenitor cells. Viable lineage-committed precursors cannot be identified until after they have expressed their mature phenotype and these precursors cannot be grown to large number while lineage is committed but still immature. Recently, we have identified stable sublines of the HL-60 human leukemia cell line which differ from each other in that they selectively differentiate to either neutrophils (UR-1-4), monocyte/macrophages (MRI), eosinophils (clones 2 and 15), or mixtures of two (clones 7 and 8) or of all three lineages (UR-1-2) when stimulated to mature with butyric acid under identical conditions. Characterization of these sublines provided evidence that the expression of lineage in HL-60 cells is a multistep process and that the lineage tendencies (lineage direction) the clones exhibited when cultured with butyric acid represent a step in that process earlier than irreversible lineage commitment but later than the multipotential wild type HL-60 cells. First, treatment of these sublines with compounds that induce differentiation of HL-60 cells to specific lineages (dimethylsulfoxide, neutrophil; 1,25-(OH)2 vitamin D3, monocyte), generally induced differentiation to the lineage associated with that inducer rather than the butyrate-associated lineage. Second, culture of neutrophil or monocyte-directed sublines in medium of elevated pH for two months leads to the development of eosinophils. Culturing the sublines first in butyric acid for variable lengths of time and switching to either DMSO or VD3 indicated that irreversible lineage commitment develops on a time course similar to the development of the commitment to mature. Markers of monocytic and eosinophilic differentiation could not be simultaneously demonstrated in single mature cells, consistent with the phenomenon of lineage fidelity. In addition, several assays were validated that could reliably classify mature HL-60 cells to their lineage. The collection of these sublines appears to constitute a model system with well-defined behavior with respect to the early events of lineage determination that can be grown to quantities sufficient for biochemical and molecular analysis. Exploring the differences between these clones may provide a new way to examine the early events of the lineage development process in myeloid cells.

Factors responsible for variable reported lineages of HL-60 cells induced to mature with butyric acid.

HL-60 is a multipotential human leukemia cell line widely used as an in vitro model to investigate myeloid differentiation. A variety of compounds can reproducibly induce these cells to differentiate towards specific lineages. However, under what appear to be similar experimental conditions, various laboratories have reported either neutrophilic differentiation or monocytic differentiation after butyric acid induction. We investigated different hypotheses to explain these dissimilar findings. First, the potential role of variable 1,25-dihydroxyvitamin D3 (VD3) concentrations in commercial fetal calf serum was assessed. Second, possible differences between laboratories inherent to the HL-60 cells themselves were explored. Lineage was assessed by morphology, histochemistry (nonspecific esterase), and neutrophil-specific (CD15) and monocyte-specific (MO2) surface antigens. We found that increasing concentrations of VD3 spanning the range reported in commercial fetal calf serum (25 to 155 pg/ml) act in synergy with butyric acid to result in higher monocyte/neutrophil ratios at the higher VD3 concentrations. Different lots of serum led to monocyte/neutrophil ratios in proportion to their VD3 concentrations. Starting with HL-60 cells obtained from different laboratories, several single-cell clones were derived which yielded either high percentages of monocytes, high percentages of neutrophils, or intermediate mixes of both cell types after induction with butyric acid. We conclude that the wide variation of VD3 concentration found in different lots of commercial fetal calf serum and intrinsic differences in HL-60 cells are two identifiable factors that can explain the discrepancies in lineage observed by different investigators after butyric acid induction of HL-60 cells.

Acute eosinophilic leukemia with a (10;11) chromosomal translocation.

We report a patient with acute eosinophilic leukemia and a translocation (10;11)(p14;q21). Clinically, the disease was characterized by extreme hypereosinophilia with most eosinophils being immature, pronounced marrow infiltration with abnormal eosinophil precursors, skin and lymphoid infiltration with leukemic eosinophils, and only a brief remission from chemotherapy. This is the second report of a patient with this cytogenetic/clinicopathological association. In our patient, t(10;11)(p14;q21) was the sole karyotypic abnormality seen in the bone marrow, both at diagnosis and relapse. Thus, acute eosinophilic leukemia with t(10;11)(p14;q21) appears to be a rare, new clinical/cytogenetic association. Because both patients with this translocation responded only briefly to chemotherapy, this chromosomal abnormality may confer a poor prognosis.

Graded increase in probability of eosinophilic differentiation of HL-60 promyelocytic leukemia cells induced by culture under alkaline conditions.

HL-60 cells differentiate primarily to eosinophils instead of neutrophils when cultured with butyric acid if they have previously been cultured under alkaline conditions (pH 7.6). To determine the nature of the process by which this occurs, a group of single-cell derived clones was produced from HL-60 cells after prolonged passage under alkaline conditions. When these clones were induced to mature with butyric acid, each clone demonstrated a characteristic proportion of mature eosinophils and neutrophils. This property was stable for multiple passages. Subclones derived from these clones also demonstrated the same probability of differentiating to an eosinophil as their parent clones. Reversion toward neutrophilic differentiation gradually occurred after several months of culture under conditions of reduced pH. The most highly directed clones demonstrated 90-95% eosinophilic differentiation and continued to differentiate primarily to eosinophils after seven months of culture at the reduced pH. Thus, in HL-60 cells, the tendency to differentiate to an eosinophil is a long-lived, heritable, continuously variable phenotype that is inducible in cells by culture under alkaline conditions. This tendency persists for prolonged periods after the alkaline conditions are removed, but may gradually revert toward neutrophilic differentiation with time.

Induction of differentiation of the human leukemia cell line, KU812.

We treated KU812 cells from different passage levels with compounds which induce differentiation of other leukemic cell lines. Early passages contained small subpopulations of basophil and erythrocyte-like cells, but later passages consisted of more uniform cells with a myelomonocytic phenotype. When cultured with 1 nM actinomycin D, the cells became nonproliferative, developed pale cytoplasm and segmented nuclei, and lost nonspecific esterase activity. Thus, KU812 cells can be induced to mature to cells with a myelomonocytic phenotype. However, their expression of previously reported basophil characteristics diminishes with continued passage, and may be a capability of only a subpopulation of the cells.

Synthesis of eosinophil-associated enzymes in HL-60 promyelocytic leukemia cells.

Eosinophils derived from HL-60 cells share many of the abnormalities of granule histochemistry and morphology frequently seen in eosinophils of patients with certain malignancies, especially those seen in acute myelomonocytic leukemia with abnormal eosinophils (FAB class M4eo). In order to understand the pathogenesis of these abnormalities, four enzymes, characteristic of the eosinophil, were studied in HL-60 promyelocytic leukemia cells at various stages of eosinophilic differentiation. Using biochemical and ultrahistochemical techniques, the following differences from normal eosinophil development were demonstrated. First, both myeloperoxidase and eosinophil peroxidase coexisted in the population of maturing HL-60 eosinophils. Second, the granules formed from the condensation of material in vacuoles which were derived from dilated segments of the endoplasmic reticulum; the role of the Golgi apparatus in processing of peroxidase appeared minimal. Third, low levels of lysophospholipase and arylsulfatase were present in the cells compared to normal eosinophils. Finally, crystallizations resembling precursor structures of Auer rods appeared in the granules of about 5% of the cells. These findings suggest that several disorders of the control of protein synthesis and processing exist in HL-60 eosinophils which may be responsible for the abnormal granule morphology and histochemistry.

Switch in differentiative response to maturation inducers of human promyelocytic leukemia cells by prior exposure to alkaline conditions.

The myeloid lineage to which HL-60 promyelocytic leukemia cells will differentiate in response to a chemical differentiation inducer can be switched by altering the pH of the growth medium. Cells passaged previously at pH 7.2 become neutrophiles, and those passaged previously at pH 7.6 become eosinophiles after 5 to 7 days of culture in the presence of 0.5 mM butyric acid. Butyric acid and its analogues are unique in that all other chemical maturation inducers tested, such as dimethyl sulfoxide and retinoic acid, promote neutrophilic differentiation regardless of the prior culture history of the cells. This suggests that lineage commitment and maturational commitment are mechanistically separate processes in this multipotential cell line and can be independently manipulated experimentally.

Development of membrane-potential responsiveness by myeloid leukemia cells during neutrophilic differentiation.

The ability of a myeloid leukemia cell line (HL-60) to undergo membrane electrical potential changes was followed during neutrophilic differentiation induced by 2 compounds. Membrane-potential changes were induced with 12-O-tetradecanoylphorbol 13-acetate (TPA) or formyl-methionyl-leucyl-phenylalanine (FMLP) and were monitored by flow cytometry. The magnitude of the membrane-potential response to TPA increased in a more uniform manner as the population of cells matured than did acquisition of mature morphology or ability to undergo the respiratory burst in response to TPA. The response to TPA and FMLP of HL-60 cells, maximally induced to differentiate by dimethylsulfoxide, closely resembled that of neutrophils. Thus, HL-60 cells may be a useful tool in the study of the relation between membrane depolarization and subsequent cellular activation.

Eosinophilic differentiation of the human promyelocytic leukemia cell line, HL-60.

HL-60 promyelocytic leukemia cells differentiated to eosinophils and eosinophilic precursors when cultured under mildly alkaline conditions (pH 7.6-7.8) for 7 d without refeeding. New cytoplasmic granules appeared blue in the least mature cells and red in the most mature cells when stained with Wright-Giemsa. The granules also stained with Luxol-fast-blue, a characteristic of eosinophil granules. Furthermore, most cells contained the eosinophil major basic protein (MBP); the Charcot-Leyden Crystal (CLC) protein (lysophospholipase), eosinophil peroxidase, acid phosphatase, and arylsulfatase were also detected in a portion of these cells. The eosinophil major basic protein was found in a high proportion of undifferentiated cells, and thus may be constituitively produced. By examining finely banded chromosomes, translocation break points were demonstrated at q22 on one chromosome 16 and at q23 on the other homologue; abnormalities in this region of the long arm of 16 are a characteristic finding in the recently described syndrome of acute myelomonocytic leukemia (AMMoL) with abnormal bone marrow eosinophils. In common with the bone marrow eosinophils in these patients, the HL-60 eosinophil granules contained chloroacetate esterase and periodic-acid Schiff (PAS) reactive material; crystalloid inclusions were rare. Therefore, the HL-60 cell line appears to be an in vitro model for eosinophilopoiesis and may be specially suited for the study of the abnormal eosinophils seen in certain malignant conditions.

Induction of neutrophilic differentiation of human promyelocytic leukemic cells by branched-chain carboxylic acid anticonvulsant drugs.

The anticonvulsant drug 1-methyl-1-cyclohexanecarboxylic acid ( MCCA ) has been shown to cause maturation of murine neuroblastoma cells in vitro at concentrations that are pharmacologically achievable. HL-60 human promyelocytic leukemia cells cultured with this drug underwent a dose-dependent decrease in growth. Similarly, neutrophilic differentiation, based on morphologic criteria and the acquisition of the ability to reduce nitroblue tetrazolium and phagocytose yeast, was observed. Valproic acid, a clinically available anticonvulsant that is chemically related to MCCA , likewise inhibited growth and promoted maturation of HL-60 cells, although only at concentrations above the recommended therapeutic blood levels. MCCA was additive in its ability to induce differentiation of HL-60 with retinoic acid, another compound that induces differentiation at pharmacologic concentrations. MCCA , or similar branched-chain fatty acids, may be useful in the treatment of human leukemia, particularly in combination with other differentiation-inducing drugs.