Lars Kjeldsen

A 25-kDa protein was found to be associated with purified human neutrophil gelatinase. Polyclonal antibodies raised against gelatinase not only recognized gelatinase but also this 25-kDa protein. Specific antibodies against the 25-kDa protein were obtained by affinity purification of the gelatinase antibodies. Immunoblotting and immunoprecipitation studies demonstrated the 135-kDa form of gelatinase to be a complex of 92-kDa gelatinase and the 25-kDa protein, and the 220-kDa form was demonstrated to be a homodimer of the 92-kDa protein, thus explaining the 220-, 135-, and 92-kDa forms characteristic of neutrophil gelatinase. The 25-kDa protein was purified to apparent homogeneity from exocytosed material from phorbol myristate acetate-stimulated neutrophils. The primary structure of the 25-kDa protein was determined as a 178-residue protein. It was susceptible to treatment with N-glycanase, and one N-glycosylation site was identified. The sequence did not match any known human protein, but showed a high degree of similarity with the deduced sequences of rat alpha 2-microglobulin-related protein and the mouse protein 24p3. It is thus a new member of the lipocalin family. The function of the 25-kDa protein, named neutrophil gelatinase-associated lipocalin (NGAL), remains to be determined.

Detection of matrix metalloproteinase (MMP) activities in the urine from patients with a variety of cancers has been closely correlated to disease status. Among these activities, the presence of a group of high molecular weight (HMW) MMPs independently serves as a multivariate predictor of the metastatic phenotype (). The identity of these HMW MMP activities has remained unknown despite their novelty and their potentially important applications in non-invasive cancer diagnosis and/or prognosis. Here, we report the identification of one of these HMW urinary MMPs of approximately 125-kDa as being a complex of gelatinase B (MMP-9) and neutrophil gelatinase-associated lipocalin (NGAL). Multiple biochemical approaches verified this identity. Analysis using substrate gel electrophoresis demonstrated that the 125-kDa urinary MMP activity co-migrates with purified human neutrophil MMP-9 x NGAL complex. The 125-kDa urinary MMP-9 x NGAL complex was recognized by a purified antibody against human NGAL as well as by a monospecific anti-human MMP-9 antibody. Furthermore, these same two antibodies were independently capable of specifically immunoprecipitating the 125-kDa urinary MMP activity in a dose-dependent manner. In addition, the complex of MMP-9 x NGAL could be reconstituted in vitro by mixing MMP-9 and NGAL in gelatinase buffers with pH values in the range of urine and in normal urine as well. Finally, the biochemical consequences of the NGAL and MMP-9 interaction were investigated both in vitro using recombinant human NGAL and MMP-9 and in cell culture by overexpressing NGAL in human breast carcinoma cells. Our data demonstrate that NGAL is capable of protecting MMP-9 from degradation in a dose-dependent manner and thereby preserving MMP-9 enzymatic activity. In summary, this study identifies the 125-kDa urinary gelatinase as being a complex of MMP-9 and NGAL and provides evidence that NGAL modulates MMP-9 activity by protecting it from degradation.

PURPOSE: Antibody-directed chemotherapy for acute myeloid leukemia (AML) may permit more treatment to be administered without escalating toxicity. Gemtuzumab ozogamicin (GO) is an immunoconjugate between CD33 and calicheamicin that is internalized when binding to the epitope. We previously established that it is feasible to combine GO with conventional chemotherapy. We now report a large randomized trial testing the addition of GO to induction and/or consolidation chemotherapy in untreated younger patients. PATIENTS AND METHODS: In this open-label trial, 1,113 patients, predominantly younger than age 60 years, were randomly assigned to receive a single dose of GO (3 mg/m(2)) on day 1 of induction course 1 with one of the following three induction schedules: daunorubicin and cytarabine; cytarabine, daunorubicin, and etoposide; or fludarabine, cytarabine, granulocyte colony-stimulating factor, and idarubicin. In remission, 948 patients were randomly assigned to GO in course 3 in combination with amsacrine, cytarabine, and etoposide or high-dose cytarabine. The primary end points were response rate and survival. RESULTS: The addition of GO was well tolerated with no significant increase in toxicity. There was no overall difference in response or survival in either induction of consolidation. However, a predefined analysis by cytogenetics showed highly significant interaction with induction GO (P = .001), with significant survival benefit for patients with favorable cytogenetics, no benefit for patients with poor-risk disease, and a trend for benefit in intermediate-risk patients. An internally validated prognostic index identified approximately 70% of patients with a predicted benefit of 10% in 5-year survival. CONCLUSION: A substantial proportion of younger patients with AML have improved survival with the addition of GO to induction chemotherapy with little additional toxicity.

In inflammatory and neoplastic disorders of the colon a defect barrier function of the mucosa may result in absorption of bacterial products from the intestinal lumen. These products may further recruit inflammatory cells and thus augment the inflammatory response. A novel lipocalin in neutrophils, neutrophil gelatinase associated lipocalin (NGAL), with the ability to bind bacterial formylpeptides, has been described and therefore it is of interest to investigate the expression of this protein in diseases of the colon. Expression of NGAL was investigated by immunohistochemistry and by mRNA in situ hybridisation in normal colon and in neoplastic and inflammatory colorectal diseases. A very high expression of NGAL was seen in colonic epithelium in areas of inflammation, both in non-malignant epithelium (diverticulitis, inflammatory bowel disease, and appendicitis) as well as in premalignant and malignant neoplastic lesions of the colon. In adenocarcinoma, the NGAL expression was especially abundant in the transitional mucosa and in the superficial ulcerated area. On the other hand, no NGAL expression could be detected in lymph node metastases from these adenocarcinomas. A weak expression of NGAL in some epithelial cells was only occasionally seen in normal colon. In conclusion, NGAL synthesis is induced in epithelial cells in inflammatory and neoplastic, colorectal diseases. NGAL may serve an important anti-inflammatory function as a scavenger of bacterial products.

PURPOSE: Treatment outcomes in younger patients with acute myeloid leukemia (AML) have improved, but optimization and new combinations are needed. We assess three combinations in induction and consolidation. PATIENTS AND METHODS: Younger untreated patients with AML (median age, 49 years; range, 0 to 73 years) were randomly allocated to two induction courses of daunorubicin and cytarabine (DA) with or without etoposide (ADE; n = 1983) or ADE versus fludarabine, cytarabine, granulocyte colony-stimulating factor, and idarubicin (FLAG-Ida; n = 1268), and to amsacrine, cytarabine, etoposide, and then mitoxantrone/cytarabine (MACE-MidAC) or high-dose cytarabine (n = 1,445) 3 g/m(2) or 1.5 g/m(2) (n = 657) in consolidation, and finally to a fifth course (cytarabine) or not (n = 227). RESULTS: Overall remission rates were similar for DA versus ADE (84% v 86%; P = .14) and ADE versus FLAG-Ida (86% v 85%; P = .7), with more course 1 remissions after FLAG-Ida (77%) reducing relapse (38% v 55%; P < .001) and improving relapse-free survival (45% v 34%; P = .01), overall and in subgroups, but with increased myelosuppression, reducing participation in the consolidation randomization. Overall outcomes were similar between MACE/MidAc and high-dose cytarabine (1.5/3.0 g/m(2)), but cytarabine required less supportive care. MACE/MidAc was superior for high-risk patients. A fifth course provided no benefit. The outcome for recipients of only two FLAG-Ida courses were not different from that with DA/ADE with consolidation. CONCLUSION: FLAG-Ida is an effective remission induction treatment, with a high complete remission rate after course 1 and reduced relapse. Consolidation with MACE/MidAc is similar overall to high-dose cytarabine, but superior in high-risk patients. Cytarabine at 1.5 g/m(2) is equivalent to a 3 g/m(2) dose. A fifth course is unnecessary. In patients receiving FLAG-Ida (two courses) and cytarabine (two courses), 8-year survival was 63% for patients with intermediate-risk and 95% for those with favorable-risk disease.