Rituximab in combination with fludarabine chemotherapy in low-grade or follicular lymphoma.

PURPOSE: To evaluate the safety and efficacy of fludarabine plus rituximab in treatment-naive or relapsed patients with low-grade and/or follicular non-Hodgkin's lymphoma. PATIENTS AND METHODS: This was an open-label, single-arm, single-center phase II study enrolling 40 patients. During the first week of the study, patients received two infusions of rituximab 375 mg/m2 administered 4 days apart. Seventy-two hours after the second infusion of rituximab, patients received the first of six cycles of fludarabine chemotherapy (25 mg/m2/d for 5 days on a 28-day cycle). Single infusions of rituximab were administered 72 hours before the second, fourth, and sixth cycles of fludarabine, and two infusions of rituximab were given 4 weeks after the last cycle of fludarabine. Treatment duration was 26 weeks. RESULTS: An overall response rate of 90% (80% complete response rate) was achieved in the intent-to-treat population. Similar response rates were seen in treatment-naive and previously treated patients. The median duration of response has not been reached at 40+ months. The median follow-up time in this study is 44 months (range, 15 to 66 months). In patients positive for the 14;18 translocation in blood and/or marrow at enrollment, molecular remission was achieved in 88% of cases, with patients remaining negative for up to 4 years to date. Hematologic toxicity was manageable, and except for a 15% incidence of herpes simplex/zoster infections, infectious complications were rare. Nonhematologic toxicities were minimal. CONCLUSION: Rituximab plus fludarabine was well tolerated and associated with an excellent complete response rate, including molecular remissions, in patients with low-grade or follicular lymphoma.

Effect of rituximab on peripheral blood stem cell mobilization and engraftment kinetics in non-Hodgkin's lymphoma patients.

Rituximab is used for in vivo tumor cell purging for non-Hodgkin's lymphoma (NHL) patients prior to autologous peripheral blood stem cell transplantation (PBSCT). However, its effects on PBSC mobilization and function are poorly understood. We compared the mobilization characteristics and engraftment kinetics of 13 NHL patients receiving and 34 NHL patients not receiving rituximab 6 months before PBSC mobilization. In the rituximab group, there was a significantly longer time to neutrophil engraftment (P=0.0466), a trend toward the need for BM harvest to supplement low-yield PBSC collections (31 vs 9%, P=0.08) and a significantly increased rate of bacteremia episodes (62 vs 26%, P=0.025). Median progression-free survival (PFS) and overall survival (OS) were significantly longer in the rituximab compared to the nonrituximab patients (P=0.049 and 0.042, respectively). However, patients in the nonrituximab group were at high risk for recurrence and expected to have shorter survival. Rituximab used within 6 months prior to collection may have a detrimental effect on PBSC mobilization and engraftment. However, rituximab is a major therapeutic breakthrough for NHL treatment and this negative effect may be offset by improved survival. Further studies are warranted in larger populations to determine the impact of rituximab on engraftment, PFS and OS.

Respiratory syncytial virus infection in the late bone marrow transplant period: report of three cases and review.

Respiratory syncytial virus (RSV) infection is an important cause of respiratory mortality in immunosuppressed patients, including bone marrow transplant (BMT) recipients. The presence of lower respiratory tract infection and infection in the pre-engraftment phase of BMT is believed to confer a poor prognosis. Three patients who underwent allogeneic BMT at our institution developed RSV pneumonia over 1 year post BMT, with the underlying disease in remission. All three were hypoxic with extensive pulmonary disease at presentation. Treatment consisted of aerosolized ribavirin and intravenous immune globulin with successful clearing of viral shedding and excellent clinical outcomes. RSV infection is probably less severe in the late post-BMT period, but needs to be considered early in the differential diagnosis of pulmonary infiltrates in this patient population.

Retrospective multivariate analysis of hepatic veno-occlusive disease after blood or marrow transplantation: possible beneficial use of low molecular weight heparin.

This retrospective cohort study of 462 consecutive adult allogeneic and autologous blood or marrow transplantation (BMT) patients compared the incidence of hepatic veno-occlusive disease (VOD) after BMT with three prophylactic regimens. Patients receiving heparin (Hep), heparin + prostaglandin E1 (Hep + PGE1) or low molecular weight heparin (LMWH) as a prophylactic VOD regimen were compared to a historical cohort receiving no VOD prophylaxis. Of 462 BMT patients, VOD was diagnosed in 22% (31 of 142) of the no prophylaxis group, 11% (11 of 104) of the Hep, 12% (13 of 110) in the Hep + PGE1 and 4% (four of 106) of the LMWH group (P = 0.0002). VOD was the primary cause of death in 20% (12 of 59). By multivariate logistic regression, independent risk factors for developing VOD were: no VOD prophylactic regimen, unrelated allogeneic BMT, Karnofsky performance score (KPS) < 80 and aspartate aminotransferase (AST) > or =50 U/l. There was no increase in the rate of death due to hemorrhagic events or VOD in any prophylaxis group compared to the control group. Prospective randomized trials of Hep vs LMWH vs placebo are warranted to assess the efficacy of heparin compounds in the prevention of VOD.

Cytokine replacement in patients with HIV-1 non-Hodgkin's lymphoma: the rationale for low-dose interleukin-2 therapy.

PURPOSE: The drastic increase in the incidence of non-Hodgkin's lymphoma in patients infected with HIV-1 is testimony to the fact that our immune system is critical for the prevention of certain malignancies. Preclinical and clinical studies were conducted to (1) gain further insight into defects in immunity that can lead to malignant transformation and (2) determine if certain immune deficiencies could be corrected by cytokines delivered at doses that result in near-physiologic concentrations in vivo. METHODS: We have used the severe combined immune deficient mouse engrafted with human peripheral blood leukocytes from healthy individuals who are seropositive for the Epstein-Barr virus to study the spontaneous development of malignant Epstein-Barr virus-positive human B-cell lymphoproliferative disorder. RESULTS: We have demonstrated in this model that, in the absence of CD4+ T cells, cytokine replacement with low-dose interleukin (IL)-2 therapy can prevent Epstein-Barr virus-positive human B-cell lymphoproliferative disorder by interacting with mouse natural killer and human CD8+ T cells. We review our clinical experience with administration of low-dose IL-2 therapy in patients with HIV-1-related cancer, noting minimal toxicity and significant immune modulation. We provide evidence that this therapy can favorably alter the type 1 cytokine profile in vivo in these patients, and improve the cellular response to infectious insults in vitro. CONCLUSION: Early clinical studies with low-dose IL-2 therapy in patients with HIV-1-related lymphoma suggest that this therapy may have a role in the prevention and treatment of this disease.

Photofrin photodynamic therapy for treatment of AIDS-related cutaneous Kaposi's sarcoma.

OBJECTIVE: Kaposi's sarcoma, the most common malignancy in AIDS patients, often presents with painful cutaneous lesions that are difficult to treat effectively despite a wide variety of therapeutic approaches. We used photodynamic therapy in an attempt to provide effective palliative treatment for this disease. METHODS: Photodynamic therapy utilizes the activation by light of a photosensitizing drug that preferentially accumulates in tumor tissue such as Kaposi's sarcoma. We enrolled 25 patients who received 1.0 mg/kg of Photofrin 48 h before exposure to 100-400 J/cm2 of 630 nm light. RESULTS: Of the 348 lesions treated, 289 were evaluable: 32.5% had complete clinical response, 63.3% had partial clinical response and 4.2% were clinical failures. There was a strong correlation between response and light dose: 54% of lesions achieved a complete clinical response at optimum light dose (> 250 J/cm2). There was no correlation of response with CD4 cell count nor was there a change in CD4 cell count post-treatment. At 400 J/cm2 full field scabbing and necrosis occurred in 90% of the treated fields. Thus, the maximum tolerated dose was determined to be 300 J/cm2. At light doses of 250 J/cm2 and below the toxicities were limited to erythema and edema in the treatment field. Forty-three biopsies were taken 0.5 h to 4 months post-treatment. These showed little change in the B and T cell infiltrates identified. Kaposi's sarcoma cells disappeared post-treatment in certain lesions. CONCLUSION: Photofrin is effective palliative treatment for HIV-associated Kaposi's sarcoma.

Natural killer cells from HIV-1+ patients produce C-C chemokines and inhibit HIV-1 infection.

Human NK cells have been shown to produce cytokines (e.g., IFN-gamma and TNF-alpha) and the chemokine macrophage inflammatory protein (MIP)-1alpha following stimulation with the combination of two monokines, IL-15 plus IL-12. The C-C chemokines MIP-1alpha, MIP-1beta, and RANTES have been identified as the major soluble macrophage-tropic HIV-1-suppressive factors produced by CD8+ T cells, which exert their action at the level of viral entry. Here, we demonstrate that monokine-activated NK cells, isolated from both normal and HIV-1+ donors, produce similar amounts of MIP-1alpha, MIP-1beta, and RANTES protein, in vitro. Further, supernatants of monokine-activated NK cells obtained from both normal donors and AIDS patients showed potent (routinely > or = 90%) suppressive activity against HIV-1 replication in vitro, compared with unstimulated control supernatants. NK cell supernatants inhibited both macrophage-tropic HIV-1(NFN-SX) and T cell-tropic HIV-1(NL4-3) replication in vitro, but not dual-tropic HIV-1(89.6). Importantly, the C-C chemokines MIP-1alpha, MIP-1beta, and RANTES were responsible only for a fraction of the HIV-1-suppressive activity exhibited by NK cell supernatants against macrophage-tropic HIV-1. Collectively these data indicate that NK cells from normal and HIV-1+ donors produce C-C chemokines and other unidentified factors that can inhibit both macrophage- and T cell-tropic HIV-1 replication in vitro. Since NK cells can be expanded in patients with HIV-1, AIDS, and AIDS malignancy in vivo, this cell type may have an important role in the in vivo regulation of HIV-1 infection.

Proglumide as a morphine adjunct in cancer pain management.

Proglumide, a cholecystokinin (CCK) antagonist, has been shown to have agonist effects at extremely low doses on both endogenous and exogenous opioid systems. To determine the effectiveness and the side effects of proglumide as an opioid agonist, a double-blind crossover study was conducted in 60 patients with cancer pain who were treated with opioid analgesics. Forty-three patients completed both treatment arms: (a) full analgesic dose plus placebo (the patient's usual analgesic dose, individualized to drug dose and route) and (b) one-half analgesic dose plus 50 mg of proglumide. An analysis of eight pain descriptors was performed to determine whether or not these treatments were associated with a difference in patients' pain perception. The level of patient anxiety differed between the two arms, but was inconsistent over time. There were no side effects detected with proglumide, as determined by clinical monitoring and patient questionnaire. No differences in pain perception were detected between the study arms. The latter finding is consistent with an augmentation of morphine analgesia, but without additional controls, the equivalency of the two arms cannot be determined with certainty. Nonetheless, this study suggests that proglumide may have use as an opioid adjunct in patients with cancer pain.

Role of transforming growth factor-beta1 in the suppressed allostimulatory function of AIDS patients.

BACKGROUND: The T-cell stimulatory function of accessory cells isolated from peripheral blood lymphocytes of AIDS patients has been reported to be suppressed. These patients also have elevated levels of the immunosuppressive factor transforming growth factor (TGF)-beta1 in their serum and plasma. OBJECTIVE: To explore the role of TGF-beta1 in the loss of accessory cell function of peripheral blood lymphocytes from AIDS patients. METHODS: Fluorescent labeled anti-TGF-beta1 and confocal microscopy were used to detect the presence of TGF-beta1 on the cell membrane of dendritic cells. To assess the role of TGF-beta1 in the inhibition of accessory cell function in AIDS, antibodies against TGF-beta1 or the TGF-beta1 type III receptor, beta-glycan, were added to a mixed lymphocyte reaction. RESULTS: TGF-beta1 was detected on the cell membrane of dendritic cells isolated from AIDS patients. The addition of blocking antibodies against either TGF-beta1 or beta-glycan restored the T-cell stimulatory function to accessory cells from these patients. CONCLUSIONS: T-cell stimulatory function was not irreversibly lost in AIDS patients. Our data suggested that beta-glycan-TGF-beta1 immunosuppressive complexes may contribute to the suppression of accessory cell function in these patients.

Ultra low dose interleukin-2 therapy promotes a type 1 cytokine profile in vivo in patients with AIDS and AIDS-associated malignancies.

This study was undertaken to determine if prolonged daily subcutaneous administration of ultra low dose IL-2 could influence the constitutive endogenous production of a type 1 (IFN-gamma) cytokine in patients with AIDS or AIDS-associated malignancies. Using a quantitative reverse transcription PCR assay, we demonstrate that daily administration of one type 1 cytokine, IL-2, for 3 mo increases significantly the constitutive endogenous gene expression of another type 1 cytokine, IFN-gamma, in vivo. The predominant source of IFN-gamma appears to be IL-2-expanded natural killer cells and CD8(+) T cells. Moreover, PBMC obtained from these patients during IL-2 therapy showed normalization of a profound deficit in IFN-gamma protein production after stimulation with extracts from infectious agents in vitro. Our data suggest that prolonged exogenous administration of a type 1 cytokine in a nontoxic fashion to patients with AIDS and AIDS-associated malignancies can enhance significantly the endogenous type 1 cytokine profile in vivo. Consequently, ultra low dose IL-2 therapy has the potential to improve the immunodeficient hosts' immune response to infectious pathogens that require IFN-gamma for clearance.

Phenotypic and functional analysis of Fas (CD95) expression in primary central nervous system lymphoma of patients with acquired immunodeficiency syndrome.

The poor prognosis associated with patients afflicted with the acquired immunodeficiency syndrome and primary central nervous system lymphoma (AIDS-PCNSL) is due in part to the intrinsic resistance of this Epstein-Barr virus (EBV)-associated tumor to conventional antineoplastic therapy. Fas (CD95) is a transmembrane protein receptor that transmits an intracellular signal leading to rapid programmed cell death following ligation with its natural ligand or anti-Fas antibodies. Fas expression and function were assessed in AIDS-PCNSL biopsy samples and in EBV+ human B-cell tumors that spontaneously developed in severe combined immune deficient (SCID) mice engrafted with human lymphocytes (hu-PBL-SCID mice). All tumors samples showed high-density surface expression of Fas by flow cytometry or immunohistochemical staining. Cells from two AIDS-PCNSL biopsy samples that did not express pan B-cell markers did not express Fas antigen. All tumors examined were susceptible to Fas-mediated apoptosis, as measured by standard assays for endonucleolytic cleavage of DNA. The response to Fas-mediated apoptosis was dependent on log-fold increases in the concentration of immobilized anti-Fas antibody, but could also be induced with a mobilized anti-Fas antibody. No evidence for intrinsic resistance to Fas-mediated apoptosis (ie, secreted or truncated forms of Fas) could be shown. Radiation-induced apoptosis of neoplastic EBV+ B cells was enhanced by activation of Fas, and prolonged exposure to interleukin-2 increased both Fas expression and Fas-induced apoptosis. As the normal brain parenchyma appears to have either low-density or absent expression of Fas, and antineoplastic therapy can be selectively delivered to the CNS with little systemic toxicity, local delivery of Fas-activating molecules could prove to be a useful component in the multimodal treatment of AIDS-PCNSL.

Immunotherapy with low-dose interleukin-2: rationale for prevention of immune-deficiency-associated cancer.

PURPOSE: Congenital, acquired, and some iatrogenically induced immune deficiencies are characterized by an increased incidence of viral-associated cancers. Preclinical and clinical studies were conducted to understand the pathogenesis of immune-deficiency-associated cancer and its response to low-dose recombinant interleukin-2 (rIL-2) therapy, with the ultimate goal of applying this or other immune therapy in the treatment or prevention of immune-deficiency-associated lymphoma. METHODS: We have used the severe combined immune-deficient (SCID) mouse engrafted with human peripheral blood lymphocytes (PBL) from healthy Epstein-Barr virus seropositive donors to study the pathogenesis of malignant B-cell lymphoproliferative disease that commonly occurs in some immune-deficient patients. In this chimeric human (hu)-PBL-SCID mouse model, administration of daily low-dose rIL-2 interacts with murine natural killer cells and human CD8+ T cells to prevent the outgrowth of human Epstein-Barr virus lymphoproliferative disease. We have utilized the information gained from this chimeric mouse model to perform a phase I study of daily, subcutaneous, low-dose rIL-2 therapy in patients with both acquired immune deficiency syndrome (AIDS) and cancer. RESULTS: Plasma concentrations of rIL-2 were achieved in vivo comparable to those seen in our hu-PBL-SCID model, in the absence of significant (grade 3) clinical toxicity or an increase in the plasma human immune deficiency virus (HIV) RNA level. Significant expansion in human cells, particularly the CD3-CD56bright natural killer cell subset, resulted after 6 weeks of therapy. Results of the hu-PBL-SCID mouse model and the phase I study have led to a national trial of low-dose rIL-2 therapy in AIDS-associated lymphoma. CONCLUSION: Daily low-dose rIL-2 therapy may be effective in treating or preventing AIDS-associated lymphoma without amplifying HIV replication.

Prolonged administration of low-dose interleukin-2 in human immunodeficiency virus-associated malignancy results in selective expansion of innate immune effectors without significant clinical toxicity.

Ten adult patients with human immunodeficiency virus (HIV)-associated malignancies (five with lymphoma and five with Kaposi's Sarcoma) were treated with a daily subcutaneous injection of interleukin-2 (IL-2) for 90 consecutive days in a phase I dose-escalation study. Seven patients had absolute CD4 counts below 200/mm3 at the time malignancy was diagnosed. Each lymphoma patient had obtained a complete or partial remission with standard chemotherapy before initiating IL-2. The daily dose of IL-2 did not change during the 90-day course of therapy. Seventeen courses of IL-2 therapy were completed at doses ranging from 0.4 x 10(6) U/m2/d to 1.2 x 10(6) U/m2/d without significant (grade III) toxicity. Two of two patients experienced grade III toxicity within 21 days of initiating IL-2 at a dose of 1.4 x 10(6) U/m2/d, but both patients subsequently completed 90 days of therapy at the maximum tolerated dose (MTD) of 1.2 x 10(6) U/m2/d. Although there were no significant increases or decreases in T-cell subsets at any dose level, there was an increase in absolute natural killer (NK) cell number at the three highest doses of IL-2 (mean percent increase 247; 95% confidence interval, 124 to 369) that was statistically significant (Wilcoxon one-sample signed rank test, P = .015). One patient developed an anti-IL-2 antibody titer that correlated with minimal NK cell expansion in vitro and in vivo. An increase in eosinophils was noted during 9 of 17 courses of IL-2 therapy without correlation to IL-2 dose, prior course of IL-2, or NK cell expansion. At the MTD, there was no consistent increase in the plasma HIV RNA level over time. Three of 10 patients had progressive disease while on study. During 50 months of IL-2 therapy, no patient was treated for an opportunistic infection. We conclude that daily low dose subcutaneous IL-2 can be self-administered safely with good compliance for prolonged periods of time to patients with HIV-associated malignancies, including those with profound immune deficiency. The majority of patients show selective expansion of innate immune effectors, ie, NK cells and/or eosinophils, in the absence of significant clinical toxicity or increased viral burden. These results suggest that low-dose IL-2 therapy should be studied further in phase II clinical trials for evidence of activity against malignancy and opportunistic infection in this patient population.

Genetic relatedness of lymphoid malignancies. Transformation of chronic lymphocytic leukemia as a model.

OBJECTIVE: Studies concerning the genetic relatedness between chronic lymphocytic leukemia and the more aggressive B-cell cancers that develop in about 10% of affected persons were reviewed. These B-cell cancers include large B-cell lymphoma (the Richter syndrome), prolymphocytic transformation, acute lymphoblastic leukemia, and multiple myeloma. Two possible relations were evaluated: development from the chronic lymphocytic leukemia clone (clonal evolution) and development of a genetically unrelated, independent second cancer. DATA SOURCES: An English-language medical literature search was done using MEDLINE (1982 to 1992) and CANCERLIT (1982 to 1992). An extensive manual search of the literature that included meeting abstracts and reports was also done. Approximately 500 articles, abstracts, and book chapters were identified; 102 were selected for detailed analysis. DATA ANALYSIS: Analysis of genetic relatedness between the two cancers considered concordance for immunoglobulin gene rearrangements, for immunoglobulin isotypes and idiotypes, and for cytogenetic abnormalities. CONCLUSIONS: In the case of large B-cell lymphoma, generally thought to arise from the chronic lymphocytic leukemia clone, approximately one half of the patients had genetically unrelated cancers. In prolymphocytic transformation, all cases studied appeared to evolve from the chronic lymphocytic leukemia clone. The few studies of acute lymphoblastic leukemia and multiple myeloma showed genetic relatedness in some cases and unrelatedness in others. These data indicate that progression to more aggressive B-cell cancers in persons with chronic lymphocytic leukemia can result from either clonal evolution or from an independent transforming event.

Clinical significance of monoclonal proteins in chronic lymphocytic leukemia.

Serum monoclonal proteins were found in 36 of 111 (32%) chronic lymphocytic leukemia (CLL) patients studied using high-resolution agarose gel electrophoresis combined with immunofixation. In contrast, using immunoelectrophoresis (IEP) and serum protein electrophoresis (SPEP), monoclonal proteins were found in 20 and 4% of CLL patients, respectively. The presence of a monoclonal protein was associated with a median survival of 63 months compared to 103 months for individuals without a monoclonal spike (p < 0.012). This was independent of clinical stage. The data also suggests that the presence of a monoclonal protein may define a group of patients at greater risk for disease progression in the low and intermediate risk groups. (Rai stages, 0, I, II). Further follow-up will be required to determine if this is statistically significant.

Renal cell carcinoma treated with continuous-infusion interleukin-2 with ex vivo-activated killer cells.

High-dose recombinant interleukin-2 (rIL-2) results in tumor responses in patients with metastatic renal cell carcinoma ranging from 9 to 31%. Continuous infusion regimens of rIL-2 may be less toxic and may result in greater in vivo lymphokine-activated killer (LAK) cell production. The current trial used a continuous infusion of rIL-2 with ex vivo LAK cells. These cells were pretreated with phenylalanine methyl ester to remove monocytes to allow cell culture at higher concentrations. Twenty-three patients were entered into the trial. Two patients had complete responses (9%) lasting 15+ and 20+ months. Four patients had partial responses (17%) of 9+, 6+, 3, and 3 months, respectively. One partial responder at 9+ months had only minimal residual retroperitoneal disease that may represent scar tissue. All responders had prior nephrectomies. All but one of the responding patients completed a full cycle of rIL-2 at the highest (starting) dose, 6 x 10(6) U/m2. This rIL-2/LAK regimen appears to be an effective therapy for metastatic renal cell carcinoma.

Interleukin-2 with ex vivo activated killer cells: therapy of advanced non-small-cell lung cancer.

A phase II study was conducted to examine the efficacy of interleukin-2 (IL-2) with lymphokine-activated killer (LAK) cells as therapy for advanced non-small-cell lung carcinoma (NSCLC). IL-2 was administered at a fixed dose of 6 x 10(6) U/M2 per day as a 24 h continuous intravenous infusion (CIV) with LAK cells. Eleven patients were entered onto this study and six were evaluable. One patient had a near complete response of 18 months duration. Only two patients were able to complete the regimen without dose reduction. This regimen was poorly tolerated with pulmonary toxicity being the major problem. The partial responder was the only patient to undergo more than one course of therapy. IL-2/LAK therapy may have activity in NSCLC and further studies are warranted in this uniformly fatal disease. However, future studies will have to incorporate less toxic IL-2 regimens.

Interleukin-2 lymphokine-activated killer cell therapy of non-Hodgkin's lymphoma and Hodgkin's disease.

We conducted a phase II study utilizing interleukin-2 (IL-2) with lymphokine-activated killer (LAK) cells as therapy for non-Hodgkin's lymphoma (NHL) and Hodgkin's disease (HD). IL-2 was given at a fixed dose of 3 x 10(6) U/m2/day administered as a 24-h continuous intravenous infusion with LAK cells. Nineteen extensively treated patients were entered and 15 were evaluable. In general, this regimen was reasonably well tolerated with mild toxicities that were rapidly reversible. Patients who completed therapy did so without dose attenuations. However, discontinuation of therapy was necessary in four patients due to atypical toxicities that were not clearly dose related. Two patients (one NHL and one HD) had partial remissions of brief duration, four had disease stabilization, and seven had progressive disease. While there were not sufficient numbers to evaluate critically any NHL or HD subtype, this regimen does not appear to have significant activity for either disease.

Multiple myeloma presenting as space-occupying lesions of the liver.

Initial myeloma presentation as discrete liver nodules is unusual. A 59-year-old man who presented with hepatomegaly and multiple space-occupying lesions in the liver and who was found to have a lambda light chain multiple myeloma with hepatic plasmacytomas is described and a review of liver involvement in multiple myeloma is presented.