c-abl is required for the development of hyperoxia-induced retinopathy.

The requirement for the nonreceptor tyrosine kinase c-abl in the pathogenesis of retinopathy of prematurity (ROP) was examined using the mouse model for ROP and c-abl-deficient mice. Hyperoxia-induced retinal neovascularization was observed in wild-type and heterozygous mice but animals that were homozygous null for c-abl did not develop a vasoproliferative retinopathy in response to hyperoxia. Two gene products, endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF), have been implicated in the pathogenesis of ROP. The mRNA expression of ET-1 and VEGF was assessed in mice maintained in normoxia and in hyperoxia-exposed mice. ET-1 mRNA levels were unchanged in wild-type mice throughout the hyperoxia treatment, suggesting that ET-1 mRNA expression is not regulated by the increase in inspired oxygen. In wild-type mice maintained in room air, VEGF mRNA levels rose threefold from postnatal day 6 (P6) to P17. When wild-type mice were treated with the hyperoxia regimen, a fivefold decrease in VEGF mRNA expression was observed from P7 to P16. However, retinal VEGF expression in hyperoxia-treated homozygous null mice did not decrease and remained at control levels. These data suggest that c-abl is required for the hyperoxia-induced retinal neovascularization and hyperoxia-induced decrease in VEGF mRNA levels.

Identification of MART-1-specific T-cell receptors: T cells utilizing distinct T-cell receptor variable and joining regions recognize the same tumor epitope.

Tumor-specific cytotoxic T lymphocytes (CTLs) can mediate tumor regression in patients with metastatic melanoma and play a central role in the immune response to cancer. The recent identification of shared melanoma antigens has raised the possibility of a limited melanoma-specific T-cell receptor (TCR) repertoire, but subsequent studies have been controversial and difficult to interpret without knowing which tumor-associated antigens (TAAs) are being recognized by specific TCRs. However, the recent cloning of several melanoma TAAs now allows for the identification of the specifically recognized TAA and its epitope. We evaluated the TCR of two clonal CD8+ CTL lines, A42 and 1E2, from two HLA-A2+ patients with metastatic melanoma. Both CTL lines were MART-1 specific, and both demonstrate reactivity to the same epitope when presented in an HLA-A2.1 context. The TCR genes of the two clones were sequenced. All of the productively rearranged A42 TCR beta chain genes were V beta 7/D beta 2.1/J beta 2.7/C beta 2; the TCR alpha chain genes were V alpha 21/J alpha 42/C alpha. The 1E2 TCR beta chain genes were V beta 3/D beta 1.1/J beta 1.1/C beta 1, and TCR alpha chains were V alpha 25/J alpha 54/C alpha. This study is the first report of TCR sequences specific for a melanoma epitope. These TCR clones may be useful for the development of more effective immunotherapies and in studies of the mechanism of T-cell recognition of tumor antigen. They also provide direct evidence that the immune system can provide more than one TCR capable of recognizing a TAA epitope.

Neutrophil-derived serine proteinases enhance membrane type-1 matrix metalloproteinase-dependent tumor cell invasion.

BACKGROUND: Matrix metalloproteinase-2 degrades a variety of basement membrane components and is essential for tumor invasion. We have previously reported that membrane type-1 matrix metalloproteinase (MT1-MMP) cooperates with neutrophil-derived serine proteinases (NDPs; elastase, cathepsin G, protease-3) to activate matrix metalloproteinase-2. We therefore hypothesized that NDPs enhance tumor-cell invasion. METHODS: Clones of human HT1080 fibrosarcoma cells transfected with MT1-MMP sense (HT-SE) or antisense CDNA (HT-AS) were used. These cells express either high (HT-SE) or extremely low levels (HT-AS) of MT1-MMP relative to nontransfected HT1080 cells (HT-WT). The cells were incubated in the presence or absence of purified NDP, with or without alpha 1-antitrypsin or the MMP inhibitor batimastat. Cell invasion was measured with the use of Boyden chambers with polycarbonate membranes coated with a reconstituted extracellular matrix. RESULTS: Under control conditions HT-WT and HT-SE cells were 4-fold more invasive than HT-AS cells. The addition of NDP increased HT-WT and HT-SE cell invasion 60% to 100% but had no effect on HT-AS cells. alpha 1-antitrypsin or batimastat did not decrease the baseline invasiveness of HT-WT and HT-SE cells; however, they abrogated the stimulatory effect of NDP. CONCLUSIONS: HT1080 cell invasion depends on MT1-MMP expression. MT1-MMP overexpression does not increase invasiveness by itself. NDPs increase invasion by MT1-MMP expressing cells by activating matrix metalloproteinase-2.

Activation of tumor cell matrix metalloproteinase-2 by neutrophil proteinases requires expression of membrane-type 1 matrix metalloproteinase.

BACKGROUND: Matrix metalloproteinase-2 (MMP-2), an enzyme involved in tumor invasion, is secreted as an inactive proenzyme and requires interaction with membrane-type 1 MMP (MT1-MMP) for activation. We have previously demonstrated that polymorphonuclear neutrophils (PMNs) release a soluble factor(s) that activates pro-MMP-2. Therefore, we tested the hypothesis that PMN-derived proteinases act in concert with MT1-MMP to activate pro-MMP-2. METHODS: Human HT-1080 cells transfected with MT1-MMP cDNA (HT-SE) or the corresponding antisense cDNA (HT-AS) or an empty vector (HT-V), which expressed differing levels of MT1-MMP, were incubated with serum-free, human PMN-conditioned medium with or without proteinase inhibitors. The culture supernatants were analyzed by gelatin zymography. RESULTS: Ht-1080 cells expressing basal (HT-V) or low levels (HT-AS) of MT1-MMP secreted MMP-2 in proenzyme from (72 kd). Ht-1080 cells with high levels of MT1-MMP (HT-SE) secreted pro MMP-2 and a 68 kd intermediate activation product. Addition of PMN-conditioned medium to either HT-SE or HT-V clones resulted in dose-dependent generation of active, 62 kd MMP-2. In contrast, when PMN-conditioned medium was added to HT-AS clones, no MMP-2 activation occurred. CONCLUSIONS: PMN-derived serine proteinases act in concert with MT1-MMP to activate proMMP-2. This finding indicates a potential role for inflammatory cells in promoting extracellular matrix breakdown during tumor invasion.

Decreasing length of stay after pancreatoduodenectomy.

HYPOTHESIS: Decreased length of stay (LOS) after pancreatoduodenectomy is due to multiple factors, including a lower complication rate and more efficient perioperative care for all patients, with and without complications. DESIGN: A retrospective review, validation cohort. SETTING: A single university hospital referral center. PATIENTS: A consecutive sample of patients undergoing pancreatoduodenectomy from January 9, 1986, to December 21, 1992 (group 1 [n = 104]) and from February 16, 1993, to November 9, 1998 (group 2 [n = 111]). INTERVENTION: Mann-Whitney test and linear [correction of logistic] regression analysis applied to clinical variables and LOS. MAIN OUTCOME MEASURES: Difference in median LOS between early and late groups and identification of factors predictive of decreased LOS. RESULTS: Total LOS decreased between the 2 groups (26 days [range, 13-117 days] vs 15 days [range, 5-61 days]; P<.001), with a decrease in preoperative (4 days [range, 0-28 days] vs 2 days [range, 0-36 days]; P<.001) and postoperative (19 days [range, 11-95 days] vs 12 days [range, 4-58 days]; P<.001) LOS (data given for group 1 vs group 2). Major complications decreased from 49% in group 1 to 25% in group 2 (P<.001). Postoperative LOS decreased for patients with (25 days [range, 15-95 days] vs 20 days [range, 8-58 days]; P = .05) and without (15 days [range, 11-47 days] vs 11 days [range, 4-55 days]; P<.001) major complications (data given for group 1 vs group 2). Multivariate analysis identified age (P = .01), pancreatic fistula (P<.001), delayed gastric emptying (P<.001), biliary complications (P<.001), operative time (P<.005), extra-abdominal infection (P<.005), use of a percutaneous stent (P = .04), and year of operation (P<.001) as independent predictors of total LOS. CONCLUSION: A reduction in complications in combination with factors leading to a streamlining of perioperative care has contributed to the decreased LOS after pancreatoduodenectomy.

Topical hepatic hypothermia attenuates pulmonary injury after hepatic ischemia and reperfusion.

BACKGROUND: Prolonged periods of hepatic ischemia are associated with hepatocellular injury and distant organ dysfunction in experimental models. Neutrophils (PMN) and tumor necrosis factor (TNF)-alpha have been implicated, mostly because of their local deleterious effects on the hepatocyte after hepatic ischemia and reperfusion (I/R) injury. We hypothesize that topical hepatic hypothermia (THH) reduces ischemia and reperfusion-induced hepatic necrosis, PMN infiltration, TNF-alpha release, and consequent acute pulmonary injury. STUDY DESIGN: Sprague-Dawley rats (250 to 300g) were evenly divided into three groups: 90 minutes of normothermic (37 degrees C) partial hepatic ischemia (normothermic I/R), 90 minutes of hypothermic (25 degrees C) partial hepatic ischemia (hypothermic I/R), and sham laparotomy (without ischemia). There were six animals in each experimental group per time point unless otherwise specified. Hepatic necrosis and PMN infiltration were evaluated and scored on hematoxylin and eosin-stained liver specimens 12 hours after reperfusion. Serum TNF-alpha levels were determined by ELISA at 0 minutes, 15 minutes, 30 minutes, 1 hour, and 12 hours postreperfusion. Pulmonary PMN infiltration and vascular permeability were measured by myeloperoxidase activity and Evans blue dye extravasation, respectively, to quantitate pulmonary injury 12 hours after reperfusion. RESULTS: Normothermic I/R results in a significant increase in TNF-alpha at 15 and 30 minutes (p < 0.005), PMN infiltration (p < 0.001), and hepatic necrosis (p < 0.001), compared with sham. Institution of THH reduced peak serum TNF-alpha levels by 54% at 15 minutes (p < 0.005) and by 73% at 30 minutes (p < 0.001) postreperfusion compared with normothermic I/R. Similarly, hepatic PMN infiltration and necrosis at 12 hours were reduced by 60% (p < 0.05) and 47% (p < 0.05), respectively. Myeloperoxidase activity and Evans blue extravasation (measures of acute lung injury) were reduced by 42% and 39%, respectively, with institution of THH compared with animals undergoing normothermic I/R (p < 0.001). CONCLUSIONS: These results demonstrate that THH protects the liver from ischemia and reperfusion-induced necrosis and PMN infiltration. In addition, THH reduces the serum levels of TNF-alpha and associated pulmonary injury. These data suggest that the ischemic liver is a potential source of inflammatory mediators associated with hepatic ischemia and reperfusion-induced pulmonary injury.

Magnetic resonance imaging with magnetic resonance cholangiopancreatography accurately predicts resectability of pancreatic carcinoma.

Accurate preoperative staging of pancreatic malignancy aids in directing appropriate therapy and avoids unnecessary invasive procedures. We evaluated the accuracy of magnetic resonance imaging (MRI) with magnetic resonance cholangiopancreatography (MRCP) in determining resectability of pancreatic malignancy. Twenty-one patients with suspected pancreatic malignancy underwent dynamic, contrast-enhanced breath-hold MRI with MRCP prior to surgical evaluation. Results of this study were correlated with operative results and pathologic findings. The sensitivity, specificity, and accuracy of MRI with MRCP in detecting a mass, determining the nature of the mass, and predicting lymph node involvement and resectability were determined. MRI with MRCP correctly identified the presence of a pancreatic mass in all 21 of these patients. Following pathologic correlation, it was determined that MRI with MRCP was 81% accurate in determining the benign or malignant nature of the pancreatic mass and 43% accurate in predicting lymph node involvement. In predicting resectability, MRI with MRCP had a sensitivity of 100%, specificity of 83%, positive predictive value of 94%, negative predictive value of 100%, and accuracy of 95%. MRI with MRCP is an accurate, noninvasive technique in the preoperative evaluation of pancreatic malignancy. Information obtained from MRI with MRCP including identification of a mass and predicting tumor resectability may be of value in staging and avoiding unnecessary invasive diagnostic procedures in patients with pancreatic cancer.

Magnetic resonance cholangiopancreatography accurately predicts the presence or absence of choledocholithiasis.

Accurate common bile duct (CBD) imaging in patients with biliary calculi is an important determinant of specific therapy. Noninvasive methods to evaluate calculi in the CBD have limited accuracy and rely mainly on ultrasonography and computed tomography. Magnetic resonance cholangiopancreatography (MRCP) is a new noninvasive modality available to evaluate the biliary system. This study was undertaken to assess the accuracy of MRCP in predicting the presence or absence of CBD stones in patients at increased risk for choledocholithiasis. The medical records of 48 patients with a final diagnosis of biliary calculous disease undergoing MRCP between November 1995 and April 1997 were retrospectively reviewed. Three groups were identified: choledocholithiasis (n = 19), gallstone pancreatitis (n 5 11), and uncomplicated cholelithiasis (n = 18). In all patients the presence or absence of CBD calculi, as determined by MRCP, was correlated with the final diagnosis obtained from endoscopic retrograde cholangiopancreatography (ERCP) (n = 19), intraoperative cholangiography (n = 6), CBD exploration (n = 13), or clinical follow-up (n = 10). Sensitivity, specificity, and accuracy of MRCP were determined. The major clinical indications for MRCP in the 48 patients ware abnormal liver function tests followed by hyperamylasemia. Twenty patients were diagnosed with CBD stones and 28 were not. MRCP correctly predicted the presence of CBD stones in 19 of 20 patients and failed to detect CBD stones in one patient with gallstone pancreatitis. MRCP incorrectly predicted the presence of CBD stones in 3 of 28 patients ultimately found to have gallstones and no CBD stones. MRCP correctly predicted the absence of CBD stones in the other 25 patients including 10 patients with gallstone pancreatitis. Overall, MRCP had a sensitivity of 95%, a specificity of 89%, and an accuracy of 92%. MRCP is an accurate, noninvasive test for evaluating the CBD duct for the presence or absence of calculi in patients suspected of having CBD stones. Our data support the use of MRCP in the preoperative evaluation of these patients as findings may influence therapeutic decisions.

Regulation of estrogen-stimulated lordosis behavior and hypothalamic progestin receptor induction by antiestrogens in female rats.

Two estrogen antagonists, CI-628 (CI) and tamoxifen (TX), were used to examine the relationship between estrogen priming of lordosis behavior and progestin receptor induction in the hypothalamus-preoptic area (HPOA) of ovariectomized female rats. Lordosis behavior was assessed by measuring lordosis quotients (LQ) in response to injection of 2 micrograms of estradiol benzoate (EB) followed 48 hr later by 500 micrograms of progesterone (P). Behavior testing began 4 hr after P injection. The effects of antiestrogens were assessed by injecting CI and TX (1-2 mg) from 0 to 48 hr prior to EB. Levels of cytosol progestin receptor in the HPOA were determined by quantifying the specific binding of 0.5 nM [3H]R5020 to cytosols from animals receiving the same EB and antiestrogen treatments used in behavioral testing. TX given concurrently with or CI given 2 hr before EB abolished both lordosis behavior and induction of HPOA progestin receptors. In contrast, CI given 12 hr prior to EB abolished lordosis but permitted a 95% elevation in the concentration of progestin binding sites in the HPOA. TX or CI given 48 hr before EB resulted in moderate levels of lordosis (mean LQs from 56 to 69) and induction of HPOA progestin receptors from 85 to 130% above noninjected controls. However, CI given 24 hr prior to EB produced less than a 40% increase in brain R5020 binding even though lordosis behavior was equivalent to that seen in the 48-hr animals (mean LQ = 53). These data indicate that the effects of antiestrogens on female sexual behavior and on the synthesis of brain progestin receptors depend on which antiestrogen is used and the time interval between administration of estrogen and antiestrogen. They also demonstrate that under some conditions estrogen induction of cytosol progestin receptors in the HPOA can be dissociated from estrogen priming of lordosis behavior in rats.

Hematopoietic stem cells and endothelial cell precursors express Tie-2, CD31 and CD45.

Early theories of tumor angiogenesis suggested that preexisting vessels surrounding the tumor were the principal source of the tumor vasculature but recent evidence suggests that endothelial progenitor cells (EPC) migrate from the marrow play an important role in developing the tumor blood supply. In a mouse model, in which the vascularization of a transplantable tumor was studied after bone marrow (BM) transplantation, we show that cells that express Tie-2, Sca-1, CD31 and CD45 function as both BM EPC and primitive hematopoietic stem cells. BM cells from transgenic mice expressing green fluorescent protein (GFP) under the control of the endothelial lineage-specific Tie-2 promoter (Tie-2 /GFP) were used to reconstitute irradiated (12 Gy) wild-type mice. Five donor BM cell populations were studied: (1) whole BM; (2) Sca-1-enriched BMC; (3) GFP/Tie-2+, Sca-1+ BMC; (4) GFP/Tie-2-, Sca-1+ BMC and (5) Sca-1-depleted BMC. After 4 weeks, the mice were injected with Tg.AC tumor cells. Three weeks later, sections from the tumors were stained for CD31 and examined for Tie-2-driven GFP expression. BM-derived endothelial cells were found only in mice transplanted with bone marrow containing populations of Tie-2+, Sca-1+ cells. As few as 3500 of these cells were sufficient to radioprotect lethally irradiated mice. Thus, we conclude that a rare subset of BMC (approximately 4 x 10(-3)%) with the putative properties of hemangioblasts have an active Tie-2 promoter. Selection of Tie-2+Sca-1+ BMC enriches for marrow-derived EPCs that participate in tumor angiogenesis and cells that can provide hematopoietic reconstitution of marrow-ablated mice.

Increased matrix metalloproteinase expression and activation following experimental acute pancreatitis.

BACKGROUND: The observation that matrix metalloproteinases (MMPs) are central to tissue remodeling and may contribute to organ failure prompted us to investigate the role of MMPs in acute pancreatitis. We hypothesize that increased expression and activation of MMP-2 and MMP-9 will correlate with organ injury following acute pancreatitis. METHODS: Acute pancreatitis was induced in five male rats by retrograde infusion of 5% sodium taurocholate into the pancreatic duct. Sham laparotomy was performed on five rats serving as a control. Pancreatitis was confirmed by histology and serum amylase levels. MMP-2 and MMP-9 activity and expression were assayed by gelatin zymography in the lungs and ascitic fluid of each animal. Lung permeability was assayed by Evans blue dye extravasation. Lung activity of MMP-2 and MMP-9 was confirmed by a specific fluorogenic MMP substrate assay. RESULTS: Lung permeability increased twofold in the animals with severe pancreatitis compared with sham. Analysis of the zymograms from lung homogenate revealed a threefold increase in active MMP-2 in severe pancreatitis compared with sham and no change in MMP-9 activity. Gelatin zymograms of peritoneal fluid from severe pancreatitis animals demonstrated increased levels of active MMP-2 and MMP-9 compared with the sham group. Increases in MMP activity were confirmed by MMP activity assay using a fluorogenic substrate. CONCLUSIONS: This study demonstrates a correlation between severity of acute pancreatitis and active MMP-2 and MMP-9 levels in the peritoneal fluid and MMP-2 activity in lung homogenate. The MMP-mediated degradation of the basement membrane offers a potential pharmacologic and therapeutic target for halting the final biologic outcome of severe pancreatitis.

Molecular mechanisms used by tumors to escape immune recognition: immunogenetherapy and the cell biology of major histocompatibility complex class I.

In this article, we explore the hypothesis that tumor cells can escape recognition by CD8+ T cells via deficiencies in antigen processing and presentation. Aspects of the molecular and cellular biology of major histocompatibility complex class I are reviewed. Evidence for histology-specific molecular mechanisms in the antigen-processing and -presentation deficiencies observed in some human and murine tumors is presented. Mechanisms identified include down-regulation of antigen processing, loss of functional beta 2-microglobulin, and deletion of specific alpha-chain alleles. Finally, we discuss studies using an antigen-presentation-deficient mouse tumor as a model for the immunogenetherapy of an antigen-presentation deficiency.

Expression of Von Willebrand factor, an endothelial cell marker, is up-regulated by angiogenesis factors: a potential method for objective assessment of tumor angiogenesis.

von Willebrand factor (vWF), a glycoprotein produced uniquely by endothelial cells and megakaryocytes, is routinely used to identify vessels in tissue sections. Vessel density in tumor specimens, as determined by immuno-histochemical staining for vWF or other endothelial cell markers, is a negative prognostic factor for many solid tumors. vWF is heterogeneously distributed throughout the vasculature, transcriptional control in response to the tissue microenvironment being responsible for local variations in endothelial cell levels of vWF. Here, we report that fibroblast growth factor-2 and vascular endothelial growth factor, potent angiogenesis inducers expressed in a variety of tumors, up-regulate expression of vWF mRNA and protein in cultured endothelial cells with a synergistic effect. Our data support the measurement of vWF mRNA in tumors to detect activated endothelium or angiogenesis. For this purpose, we developed a semi-quantitative RT-PCR for vWF mRNA. Preliminary results obtained with specimens from colon carcinoma and the corresponding normal colonic mucosa showed higher vWF mRNA levels in most tumors than in their normal counterparts. The differences in vWF mRNA levels were much larger than the differences in vessel counts between a tumor and the corresponding normal mucosa, indicating that high vWF mRNA levels in tumors may indeed be an early sign of activation of the endothelium. The rapidity, objectivity, sensitivity and specificity of this technique make it suitable for routine clinical application to identify aggressive, highly angiogenic tumors.

Recognition of neuroectodermal tumors by melanoma-specific cytotoxic T lymphocytes: evidence for antigen sharing by tumors derived from the neural crest.

Melanomas from different patients have been shown to express shared tumor antigens, which can be recognized in the context of the appropriate MHC class I molecules by cytolytic T cells. To determine if T-cell-defined melanoma antigens are expressed on other tumors of neuroectodermal origin, four melanoma-specific cytotoxic T lymphocyte (CTL) cultures derived from tumor-infiltrating lymphocytes (TIL) were tested for lysis of a panel of 23 HLA-A2+ neuroectodermal tumor cell lines of various histologies, including retinoblastoma (1), neuroblastoma (8), neuroepithelioma (6), astrocytoma (2), neuroglioma (1), and Ewing's sarcoma (5). Low expression of MHC class I and/or ICAM-1 molecules was found on 22 of 23 neuroectodermal tumor lines, and could be enhanced by treatment with interferon gamma (IFN gamma). Following IFN gamma treatment, three Ewing's sarcoma lines were lysed by at least one melanoma TIL culture, and levels of lysis were comparable to melanoma lysis by these TIL. Lysis could be inhibited by monoclonal antibodies directed against MHC class I molecules and against CD3, indicating specific immune recognition of tumor-associated antigens. None of the other neuroectodermal tumors tested were lysed by TIL, but they could be lysed by non-MHC-restricted lymphokine-activated killer cells. This demonstration of immunological cross-reactivity between melanomas and Ewing's sarcomas, two tumors of distinct histological types with a common embryonic origin, has implications for the developmental nature of these CTL-defined tumor antigens. It also raises the possibility that specific antitumor immunotherapies, such as vaccines, may be reactive against more than one form of cancer.

Endoscopic biliary drainage before pancreaticoduodenectomy for periampullary malignancies.

Despite decreased operative mortality, pancreaticoduodenectomy (PD) remains a formidable operation with substantial morbidity. We have evaluated the influence of preoperative endoscopic biliary drainage (EBD) on morbidity after PD for malignant biliary obstruction by retrospectively reviewing the medical records of 182 patients undergoing PD between April 1985 and August 1996. Of 52 study patients with malignant obstructive jaundice, 22 underwent preoperative EBD, and 30 were not drained. Eighty-three patients were excluded for bilirubin levels less than 5 mg/dl, 43 had other biliary drainage, and 4 had jaundice with benign pathology. Preoperative, intraoperative, and postoperative factors were compared. The two groups were well matched for clinical presentation and operative characteristics except for lower preoperative values of liver chemistries in patients undergoing EBD. Length of postoperative hospitalization for patients undergoing EBD was 13.5 days, compared with 19 days for patients who were not drained (p = 0.02). Patients who were not drained tended to have more overall complications (p = 0.054). Multivariate analysis revealed time to regular diet (p < 0.0001) and no preoperative drainage (p = 0.04) to be independent factors significantly increasing the length of hospitalization. Endoscopic biliary drainage before PD significantly reduced the length of postoperative hospitalization and was associated with less postoperative morbidity. Further studies, including cost analysis, are warranted.

Activation of progelatinase A (MMP-2) by neutrophil elastase, cathepsin G, and proteinase-3: a role for inflammatory cells in tumor invasion and angiogenesis.

Gelatinase A (MMP-2), a matrix metalloproteinase (MMP) involved in tumor invasion and angiogenesis, is secreted as an inactive zymogen (proMMP-2) and activated by proteolytic cleavage. Here we report that polymorphonuclear neutrophil (PMN)-derived elastase, cathepsin G, and proteinase-3 activate proMMP-2 through a mechanism that requires membrane-type 1 matrix metalloproteinase (MT1-MMP) expression. Immunoprecipitation of human PMN-conditioned medium with a mixture of antibodies to elastase, cathepsin G, and proteinase-3 abolished proMMP-2 activation, whereas individual antibodies were ineffective. Incubation of HT1080 cells with either purified PMN elastase or cathepsin G or proteinase-3 resulted in dose-and time-dependent proMMP-2 activation. Addition of PMN-conditioned medium to MT1-MMP expressing cells resulted in increased proMMP-2 activation and in vitro invasion of extracellular matrix (ECM), but had no effect with cells that express no MT1-MMP. MMP-2 activation by PMN-conditioned medium or purified elastase was blocked by the elastase inhibitor alpha(1)-antitrypsin but not by Batimastat, an MMP inhibitor, showing that elastase activation of MMP-2 is not mediated by MMP activities. The PMN-conditioned medium-induced increase in cell invasion was blocked by Batimastat as well as by alpha(1)-antitrypsin, showing that PMN serine proteinases trigger a proteinase cascade that entails proMMP-2 activation: this gelatinase is the downstream effector of the proinvasive activity of PMN proteinases. These findings indicate a novel role for PMN-mediated inflammation in a variety of tissue remodeling processes including tumor invasion and angiogenesis.

Locus-specific analysis of human leukocyte antigen class I expression in melanoma cell lines.

Surface expression of human leukocyte antigen (HLA) class I antigens on melanoma lines was evaluated by locus-specific monoclonal antibodies (mAbs) with three different techniques: Fluorescence-activated cell sorting (FACS), immunohistochemistry with cytospin preparation (ICP), and complement-mediated cytotoxicity (CMC). Eleven HLA class I-expressing cell lines developed from metastases were used. Specific expression of HLA loci was examined under routine culture conditions and after 48-h incubation in interferon-gamma (IFN-gamma; 500 U/ml). Loss of allelic expression was seen in one line (586-MEL): Products of genes coding for HLA-A29 and -B44, in strong linkage disequilibrium, were not detectable. HLA-A antigens were consistently detected by all methodologies and minimally affected by pretreatment with IFN-gamma. HLA-B antigens were detectable in 8 of 11 lines by ICP and 3 of 11 lines by CMC. By FACS the supratypic specificity HLA-Bw6 was expressed at low levels in most lines (mean fluorescence 47.2 +/- 13.4 and rose to 259.8 +/- 45.9 after incubation with IFN-gamma; p < 0.001). HLA-Cw antigen detection by CMC correlated with HLA-B (p < 0.01), suggesting that down-regulation and sensitivity to IFN-gamma are shared by the two loci. This low expression of the HLA-B antigens may play a role in the evasion of the host immune response and its up-regulation may be useful in allowing tumor antigen recognition.

HLA associations in the antitumor response against malignant melanoma.

In this study we analyzed the human leukocyte antigen (HLA) pattern of North American Caucasian patients with metastatic melanoma as compared with the North American Caucasian (NAC) population. We also investigated whether the HLA type of melanoma patients had an effect on their tolerance and response to interleukin-2 (IL-2)-based therapy. Four hundred twelve serologic phenotypes of Caucasian melanoma patients referred to the National Cancer Institute, National Institutes of Health, from February 1989 through December 1993 were collected by typing the patient's peripheral blood lymphocytes. Furthermore, 74 melanoma patients were typed for HLA class II by high-resolution sequence specific primer-polymerase chain reaction. Response rate and treatment-related toxicity in those patients receiving IL-2-based treatment (N = 272) were compared with HLA serologic types. The frequency of four HLA-B alleles was significantly different in the melanoma compared with the NAC population: of these, HLA-B5, -B8, and -B15 had a frequency falling between the NAC and the Northern European population. No other significant differences between melanoma patients and NAC population were noted for other HLA loci. A correlation was noted between HLA-DR3 and -DR4 alleles and decreased tolerance to IL-2, whereas homozygosity for HLA-DR decreased the chance of response. There were no significant associations between HLA type and response. It is unlikely that the associations noted between some HLA-B alleles and melanoma bear significantly on the etiology of the disease. The differences seen between American melanoma patients and the NAC population are probably best explained by geographical ancestry. The association between HLA-DR and tolerance to IL-2 therapy noted in this study may offer insight toward the understanding of mechanisms regulating the cascade of events after the systemic administration of IL-2.