Urokinase receptor antibody can reduce tumor volume and detect the presence of occult tumor metastases in vivo

The serine protease urinary plasminogen activator or urokinase (uPA), produced in abundance by many malignancies, plays a key role in tumor cell invasion and metastasis. uPA is localized within the malignant cell milieu via its cell surface receptor [uPA receptor (uPAR)], which is expressed by tumor and tumor-associated cells. In the present study, we have used a syngeneic model of rat breast cancer to directly evaluate the role of uPAR as a diagnostic and therapeutic target in metastatic breast cancer. A polyclonal antibody against the ligand-binding NH(2)-terminal domain of rat uPAR (ruPAR) was developed. This antibody recognizes ruPAR by both immunofluorescence and Western blot analysis. Recombinant ruPAR and ruPAR IgG displaced the binding of (125)I-labeled ruPAR IgG to rat prostate cancer cells (Dunning R3227 Mat Ly Lu) and breast cancer cells (Mat B-III) overexpressing ruPAR (Mat B-III-uPAR). ruPAR IgG also blocked the invasive capacity of these tumor cells in a dose-dependent manner. Mat B-III-uPAR cells were inoculated s.c. into the mammary fat pad of syngeneic female Fischer rats. On day 10 after tumor cell inoculation, animals were injected with (125)I-labeled preimmune or ruPAR IgG and then sacrificed at timed intervals. Maximum (125)I uptake was observed in primary tumors and in tissues commonly affected by tumor metastases (liver, spleen, lungs, and lymph nodes) at 12 h. Injection of (125)I-labeled preimmune or ruPAR IgG into normal non-tumor-bearing animals resulted in minimal basal levels of uPAR expression and established the specificity of the ruPAR IgG. Similar results were obtained by Northern blot and PCR analysis of mRNA isolated from tissues of normal and tumor-bearing animals. To evaluate the effectiveness of this antibody in tumor progression, ruPAR IgG (50-100 microg/day) was injected s.c. for 7 days (day 1-7) at the site of tumor cell inoculation (mammary fat pad), and animals were sacrificed at various time points for evaluation of tumor growth and metastases. Animals receiving ruPAR IgG showed a marked decrease in tumor growth and metastases as compared with control tumor-bearing animals receiving the same dose of preimmune rabbit IgG. Histological analysis of experimental primary tumors showed marked tumor necrosis that was due to increased tumor cell apoptosis as determined by terminal deoxynucleotidyl transferase-mediated nick end labeling assay. Together, these studies demonstrate the ability of anti-uPAR antibody to decrease tumor volume and detect the presence of microscopic occult tumor metastases in malignancies where uPA/uPAR play a key role in tumor progression.     

Impact of alterations affecting the p53 pathway in bladder cancer on clinical outcome, assessed by conventional and array-based methods.

This study was designed to define the potential clinical relevance of identifying alterations affecting p53 pathway in bladder cancer and to test a new, low-cost, high-throughput, and array-based TP53 sequencing technology. Tumor samples from 140 evaluable patients with bladder cancer were analyzed with two methods to detect TP53 gene mutations, including single-stranded conformational polymorphism followed by direct sequencing and an oligonucleotide array-based sequencing method. Immunohistochemistry was used to assess patterns of expression of p53, p21/WAF1, and mdm2. Median follow-up time was 27.6 months. Results from the above analyses were correlated with clinicopathological parameters and outcome. Combining the mutation-detection assays, 79 cases (56.4%) were found to harbor TP53 gene mutations. Direct sequencing identified 66 point mutations and five frameshift mutations. The p53 oligonucleotide array detected 65 point mutations and four splice site mutations in different exons but missed all five frameshift mutations. p53 nuclear overexpression was observed in 71 cases (50.7%), lack of p21 nuclear expression was found in 81 cases (57.9%), and mdm2 nuclear overexpression was seen in 64 cases (45.7%). In multivariate analysis, 17 patients (12.1%) had an altered p53 pathway, defined by the detection of mutant TP53 and/or p53 nuclear overexpression, loss of p21 nuclear expression, and mdm2 nuclear overexpression, and exhibited the worst clinical outcome in the observation period (P = 0.015), and it appears to be a significant prognostic factor associated with patient survival.     

Phase I trial of intravesical gemcitabine in bacillus Calmette-Guérin-refractory transitional-cell carcinoma of the bladder.

PURPOSE: The aim of this phase I study was to determine the safety and toxicity profile of gemcitabine administered as an intravesical agent in patients with transitional-cell carcinoma (TCC) of the bladder. PATIENTS AND METHODS: Patients with superficial bladder cancer refractory to intravesical bacillus Calmette-Guérin (BCG) therapy and refusing a cystectomy were considered eligible for the trial. Gemcitabine was given in the bladder for 1 hour twice weekly in 100 mL sodium chloride for a total of six treatments. After a 1-week break, a second course of six treatments over 3 weeks was given, followed by response assessment. Four dose levels were explored: 500 mg, 1,000 mg, 1,500 mg, and 2,000 mg. RESULTS: Eighteen patients completed therapy: three at 500 mg, six at 1,000 mg, three at 1,500 mg, and six at 2,000 mg. No grade 3 or 4 toxicity was observed at 500 mg. At 1,000 mg, three patients developed hematuria and one had a skin reaction resembling grade 3 hand-foot syndrome. Three patients at 1,500 mg had no grade 3 or 4 toxicity. Of six patients at 2,000 mg, one had grade 3 thrombocytopenia and neutropenia without infection. Seven patients had a complete response (negative cytology and posttreatment biopsy), and four patients had a mixed response (negative bladder biopsy but positive cytology). CONCLUSION: Gemcitabine has substantial activity as an intravesical agent in BCG-refractory TCC and warrants further investigation. Therapy given twice weekly was associated with minimal bladder irritation and tolerable myelosuppression. The recommended phase II dose for twice-weekly therapy is 2,000 mg.     

Lead nitrate induced apoptosis in alveolar macrophages from rat lung

In this work we have attempted to characterize the programmed cell death (apoptosis) in alveolar macrophages exposed to various concentrations of lead nitrate. It was found that after 3 h of exposure a significant increase in superoxide anion production was observed, i.e. the number of trypan blue - exculding cells, was unchanged (< or = 95%) with any dose of lead employed. Agarose gel electrophoresis and diphenylamin reaction analysis revealed the occurrence of internucleosomal DNA fragmentation evaluated using cytological analysis by fluorescence dyes, suggesting that lead nitrate at low concentrations and short periods of exposure leads macrophages into apoptosis. However, time course studies showed that beyond 3 h, toxicity occurs, which could be attenuated by phosphodiesterase inhibitors, such as caffeine, suggesting a possible mechanism involving cAMP.     

Anti-human leukocyte antigen antibodies are associated with restenosis after percutaneous coronary intervention for cardiac allograft vasculopathy

BACKGROUND: Percutaneous coronary intervention (PCI) to palliate cardiac allograft vasculopathy (CAV) has been associated with high restenosis rates, possibly related to increased inflammation associated with this disease. Whether markers of immunologic rejection are associated with restenosis in this population is unknown. The goal of the study was to determine the predictors of restenosis after PCI for CAV. METHODS: Records were reviewed retrospectively from a single, high-volume cardiac transplant center. Clinical, angiographic, and immunologic data were collected on all patients postorthotopic heart transplantation (OHT) that had subsequent PCI. Restenosis was defined as greater than 50% stenosis at the previous intervention site. RESULTS: PCI was successfully performed on 62 de novo lesions in 40 patients an average of 6.8+/-3.9 years after OHT. Angiographic follow-up data was available for 79%, with an average follow-up of 1.54+/-1.22 years. The 1-year restenosis rate was 49% (64% for balloon percutaneous transluminal coronary angioplasty and 33% for coronary stenting [P=0.09 for difference]). The frequency of immunoglobulin (Ig)G antibody to major histocompatibility complex (MHC) class I antigen was highly associated with risk of restenosis (hazard ratio [HR] 11.33, P=0.01). Greater stenosis severity and smaller target vessel diameter were also predictors of restenosis as in the nontransplant population. CONCLUSIONS: The findings suggest that in patients postPCI for CAV, humoral allo-immunity may contribute to restenosis and that IgG antibodies to MHC class I antigen may help predict the risk of restenosis after PCI in this population.     

Evidence for C-Reactive Protein's Role in (CRP) Vascular Disease: Atherothrombosis,Immuno-Regulation and CRP

For clinicians plasma C-reactive protein (CRP) levels are the only widely available tests that provide a tangible link between inflammation and atherosclerosis. New AHA/CDC joint guidelines from 2002-03 now include the measurement of CRP as a class IIa recommendation for stratifying patients with known cardiovascular disease (CVD) at a moderate (10-20%) 10-year event risk and a class IIb recommendation for patients without known CVD [1]. While the association of CRP and atherosclerosis is by now accepted, the molecular biology behind the association is evolving rapidly into a fascinating story. While some of the story remains obscure, this review aims to bridge the clinical and basic science and identify what is known about the role of this ancient molecule in atherosclerosis. The review covers CRP's interaction with atherosclerosis' major ingredients and cell types including the endothelium, monocytes and neutrophils, lipoproteins and the complement system. Taken together, the clinical and basic science leave the tantalizing impression that CRP has a fundamental role in atherogenesis, and hint at a more complex immunomodulatory effect which transforms the acute inflammatory response to vascular injury into the chronic inflammation seen in atherosclerosis.     

Minocycline inhibits smooth muscle cell proliferation, migration and neointima formation after arterial injury

The tetracyclines are antimicrobials that also inhibit expression of certain matrix metalloproteinases (MMPs). We conducted a series of experiments to determine if minocycline could inhibit MMP expression and limit human aortic smooth muscle cell (SMC) proliferation and migration. Analysis of SMC proliferation was performed after cells were grown in minocycline-incubated media. SMC migration activity was assayed in a micro-Boyden chamber. Western blotting revealed that minocycline reduced SMC production of MMP-2 in a dose dependent manner. Increasing doses of minocycline progressively reduced SMC proliferation to 49% of control values and limited SMC migration to 15% of control. When administered to rats with balloon injured carotid arteries, intraperitoneal doses of minocycline (70-100 mg/kg) reduced neointima formation by 76%, but were associated with liver toxicity. Higher doses were lethal and lower doses were ineffective. Minocycline, applied to injured arteries in a pluronic gel with a low pH, was also ineffective. In summary, minocycline lowers MMP-2 expression, reduces SMC proliferation and migration, and inhibits neointimal hyperplasia, but its efficacy is limited by systemic toxicity.     

Prostate secretory protein PSP-94 decreases tumor growth and hypercalcemia of malignancy in a syngenic in vivo model of prostate cancer

Prostate cancer is a common malignancy affecting men, which is often associated with skeletal metastases resulting in significant morbidity and mortality. In this hormone-dependent cancer, low levels of a prostate secretory protein of 94 amino acids (PSP-94) are associated with advanced disease stage. In the current study, we have examined the effect of PSP-94 on prostate cancer growth and experimental metastases to the skeleton. For these studies, MatLyLu rat prostate cancer cells were transfected with full-length cDNA encoding parathyroid hormone-related protein [PTHrP (MatLyLu-PTHrP cells)], which is known to be the major pathogenetic factor for malignancy-associated hypercalcemia. MatLyLu-PTHrP cells were inoculated s.c. into the right flank or via intracardiac route into the left ventricle of syngeneic male Copenhagen rats. Intracardiac inoculation of MatLyLu cells routinely results in the development of tumors in the lumbar vertebrae, resulting in hind-limb paralysis. Animals were infused with different doses of PSP-94 (0.1, 1.0, and 10.0 micro g/kg/day) starting on the day of tumor cell inoculation. Time of hind-limb paralysis and tumor volume were determined, and comparison was made between PSP-94-treated animals and control animals receiving vehicle alone. At the end of the study, animals were sacrificed, and plasma calcium, plasma PTHrP, and tumor PTHrP levels were determined. Whereas the highest dose of PSP-94 caused a modest but statistically significant delay in the development of hind-limb paralysis, a marked dose-dependent decrease in primary tumor volume was seen in experimental animals receiving PSP-94 due to its ability to promote tumor cell apoptosis. Furthermore, whereas control animals routinely developed hypercalcemia due to PTHrP production, treatment with PSP-94 led to a near normalization of plasma calcium and a marked reduction in PTHrP production as determined by radioimmunoassay and immunohistochemistry. Collectively, these results demonstrate the ability of PSP-94 to be an effective treatment modality for prostate cancer, where decrease in plasma PTHrP and calcium levels can serve as useful biochemical markers for monitoring the efficacy of this novel antitumor agent.     

Amelioration of immune-mediated experimental colitis: tolerance induction in the presence of preexisting immunity and surrogate antigen bystander effect

Oral tolerance is a recognized procedure for induction of antigen-specific peripheral immune hyporesponsiveness. Recently, it has been shown that oral tolerance can be used to prevent experimental colitis. The aim of this study was to test whether induction of oral tolerance toward proteins extracted from inflammatory and noninflammatory colons can alleviate preexisting experimental colitis. Colitis was induced in BALB/c mice by intracolonic instillation of 2,4,6-trinitrobenzenesulfonic acid (TNBS). Mice received five oral doses of colonic proteins extracted from TNBS-induced colitis or normal colons, before, or 7 days after colitis was induced. Standard clinical, macroscopic, and microscopic scores were used for colitis assessment. Serum interferon gamma (IFNgamma) and interleukin (IL)4 levels were measured by enzyme-linked immunosorbent assay. Feeding of colitis- or normal colon-extracted proteins before, or following colitis induction, ameliorated colonic inflammation as shown by decreased diarrhea, increased body weight, reduction of colonic ulcerations, intestinal and peritoneal adhesions, wall thickness, and edema. Histological parameters for colitis were markedly improved in tolerized animals, and there was a significant reduction in inflammatory response and mucosal ulcerations. Tolerized mice developed an increase in IL4 and a decrease in IFNgamma serum levels. The results show that induction of oral tolerance to colitis- or normal colon-extracted proteins down-regulated preexisting anticolon immune response, thereby ameliorating experimental colitis. Tolerance induction was mediated via a shift from a proinflammatory T helper (Th)1 to an anti-inflammatory Th2 immune response.