Analytical validation of a multi-cancer early detection test with cancer signal origin using a cell-free DNA-based targeted methylation assay.

The analytical validation is reported for a targeted methylation-based cell-free DNA multi-cancer early detection test designed to detect cancer and predict the cancer signal origin (tissue of origin). A machine-learning classifier was used to analyze the methylation patterns of >105 genomic targets covering >1 million methylation sites. Analytical sensitivity (limit of detection [95% probability]) was characterized with respect to tumor content by expected variant allele frequency and was determined to be 0.07%-0.17% across five tumor cases and 0.51% for the lymphoid neoplasm case. Test specificity was 99.3% (95% confidence interval, 98.6-99.7%). In the reproducibility and repeatability study, results were consistent in 31/34 (91.2%) pairs with cancer and 17/17 (100%) pairs without cancer; between runs, results were concordant for 129/133 (97.0%) cancer and 37/37 (100%) non-cancer sample pairs. Across 3- to 100-ng input levels of cell-free DNA, cancer was detected in 157/182 (86.3%) cancer samples but not in any of the 62 non-cancer samples. In input titration tests, cancer signal origin was correctly predicted in all tumor samples detected as cancer. No cross-contamination events were observed. No potential interferent (hemoglobin, bilirubin, triglycerides, genomic DNA) affected performance. The results of this analytical validation study support continued clinical development of a targeted methylation cell-free DNA multi-cancer early detection test.

Rare autosomal trisomies, revealed by maternal plasma DNA sequencing, suggest increased risk of feto-placental disease.

Whole-genome sequencing (WGS) of maternal plasma cell-free DNA (cfDNA) can potentially evaluate all 24 chromosomes to identify abnormalities of the placenta, fetus, or pregnant woman. Current bioinformatics algorithms typically only report on chromosomes 21, 18, 13, X, and Y; sequencing results from other chromosomes may be masked. We hypothesized that by systematically analyzing WGS data from all chromosomes, we could identify rare autosomal trisomies (RATs) to improve understanding of feto-placental biology. We analyzed two independent cohorts from clinical laboratories, both of which used a similar quality control parameter, normalized chromosome denominator quality. The entire data set included 89,817 samples. Samples flagged for analysis and classified as abnormal were 328 of 72,932 (0.45%) and 71 of 16,885 (0.42%) in cohorts 1 and 2, respectively. Clinical outcome data were available for 57 of 71 (80%) of abnormal cases in cohort 2. Visual analysis of WGS data demonstrated RATs, copy number variants, and extensive genome-wide imbalances. Trisomies 7, 15, 16, and 22 were the most frequently observed RATs in both cohorts. Cytogenetic or pregnancy outcome data were available in 52 of 60 (87%) of cases with RATs in cohort 2. Cases with RATs detected were associated with miscarriage, true fetal mosaicism, and confirmed or suspected uniparental disomy. Comparing the trisomic fraction with the fetal fraction allowed estimation of possible mosaicism. Analysis and reporting of aneuploidies in all chromosomes can clarify cases in which cfDNA findings on selected "target" chromosomes (21, 18, and 13) are discordant with the fetal karyotype and may identify pregnancies at risk of miscarriage and other complications.

Fetal aneuploidy screening with cell-free DNA in late gestation.

OBJECTIVE: The aim of this study was to evaluate clinical use of NIPT at gestational ages of 23 weeks and above. METHODS: A cohort of 5579 clinical patients with singleton gestations of 23 weeks or greater submitting a blood sample for NIPT in an 18-month period were selected for this study. Clinical outcomes were requested for samples with NIPT results indicating fetal aneuploidy and compared with NIPT findings to confirm concordance or discordance. RESULTS: A review of clinical indications revealed that a significantly (p < 0.0001) larger proportion of late-gestation samples indicated abnormal ultrasound findings with or without other indications, 6.2% and 42.1%, compared with early-gestation samples, 1.8% and 6.0%, respectively. Of 5372 reported late-gestation samples, 151 (2.8%) were reported as aneuploidy detected or suspected. In late-gestation samples, the overall observed positive predictive value (PPV) for NIPT was 64.7%, with an observed PPV of 100% in the subset of cases with multiple clinical indications including abnormal ultrasound findings. CONCLUSIONS: NIPT is a highly accurate prenatal screening option for women after 23 weeks of gestation. Women who presented for NIPT in the latter stages of pregnancy more frequently specified clinical indications of abnormal ultrasound findings than women who entered screening earlier in pregnancy.

Noninvasive prenatal testing in the general obstetric population: clinical performance and counseling considerations in over 85 000 cases.

OBJECTIVE: The primary goal of this study was to provide clinically relevant information for appropriate patient counseling. METHOD: Demographics and test metrics were reviewed for 86 658 clinical cases. Outcome information was requested for samples reported as aneuploidy detected or suspected for chromosomes 21, 18, or 13; voluntary outcome reporting was encouraged for all discordant outcomes. RESULTS: Of 86 658 cases, 85 298 (98.4%) met inclusion criteria for result reporting. Of the 1360 (1.6%) cancellations, only 101 (0.1%) were for technical reasons. Average time to result was 3.3 business days. Aneuploidy was detected or suspected in 2142 (2.5%) samples. For aneuploidy detected cases with known clinical outcomes, the overall positive predictive value (PPV) was 83.5% (608/728); observed PPVs for trisomies 21, 18, and 13 ranged from 50.0 to 92.8%. As individual PPVs are determined by a patient's prior risk, we developed a chart for counseling patients on positive predictive value based on maternal age. CONCLUSION: This large-scale report reinforces that noninvasive prenatal testing is a highly accurate screen for fetal aneuploidy in the general obstetric population. Test improvements have facilitated a reduction in failure rates, time to result, and borderline results/unclassifiable results. We have developed a positive predictive value counseling tool to ensure appropriate patient education, counseling, and clinical utilization.

Noninvasive Prenatal Testing and Incidental Detection of Occult Maternal Malignancies.

IMPORTANCE: Understanding the relationship between aneuploidy detection on noninvasive prenatal testing (NIPT) and occult maternal malignancies may explain results that are discordant with the fetal karyotype and improve maternal clinical care. OBJECTIVE: To evaluate massively parallel sequencing data for patterns of copy-number variations that might prospectively identify occult maternal malignancies. DESIGN, SETTING, AND PARTICIPANTS: Case series identified from 125,426 samples submitted between February 15, 2012, and September 30, 2014, from asymptomatic pregnant women who underwent plasma cell-free DNA sequencing for clinical prenatal aneuploidy screening. Analyses were conducted in a clinical laboratory that performs DNA sequencing. Among the clinical samples, abnormal results were detected in 3757 (3%); these were reported to the ordering physician with recommendations for further evaluation. EXPOSURES: NIPT for fetal aneuploidy screening (chromosomes 13, 18, 21, X, and Y). MAIN OUTCOMES AND MEASURES: Detailed genome-wide bioinformatics analysis was performed on available sequencing data from 8 of 10 women with known cancers. Genome-wide copy-number changes in the original NIPT samples and in subsequent serial samples from individual patients when available are reported. Copy-number changes detected in NIPT sequencing data in the known cancer cases were compared with the types of aneuploidies detected in the overall cohort. RESULTS: From a cohort of 125,426 NIPT results, 3757 (3%) were positive for 1 or more aneuploidies involving chromosomes 13, 18, 21, X, or Y. From this set of 3757 samples, 10 cases of maternal cancer were identified. Detailed clinical and sequencing data were obtained in 8. Maternal cancers most frequently occurred with the rare NIPT finding of more than 1 aneuploidy detected (7 known cancers among 39 cases of multiple aneuploidies by NIPT, 18% [95% CI, 7.5%-33.5%]). All 8 cases that underwent further bioinformatics analysis showed unique patterns of nonspecific copy-number gains and losses across multiple chromosomes. In 1 case, blood was sampled after completion of treatment for colorectal cancer and the abnormal pattern was no longer evident. CONCLUSIONS AND RELEVANCE: In this preliminary study, a small number of cases of occult malignancy were subsequently diagnosed among pregnant women whose noninvasive prenatal testing results showed discordance with the fetal karyotype. The clinical importance of these findings will require further research.

Fetal sex chromosome testing by maternal plasma DNA sequencing: clinical laboratory experience and biology.

OBJECTIVE: To describe the clinical experience with noninvasive prenatal testing for fetal sex chromosomes using sequencing of maternal plasma cell-free DNA in a commercial laboratory. METHODS: A noninvasive prenatal testing laboratory data set was examined for samples in which fetal sex chromosomes were reported. Available clinical outcomes were reviewed. RESULTS: Of 18,161 samples with sex chromosome results, no sex chromosome aneuploidy was detected in 98.9% and the fetal sex was reported as XY (9,236) or XX (8,721). In 4 of 32 cases in which the fetal sex was reportedly discordant between noninvasive prenatal testing and karyotype or ultrasonogram, a potential biological reason for the discordance exists, including two cases of documented co-twin demise, one case of a maternal kidney transplant from a male donor, and one case of fetal ambiguous genitalia. In the remaining 204 samples (1.1%), one of four sex chromosome aneuploidies (monosomy X, XXX, XXY, or XYY) was detected. The frequency of false positive results for sex chromosome aneuploidies is a minimum of 0.26% and a maximum of 1.05%. All but one of the discordant sex chromosome aneuploidy results involved the X chromosome. In two putative false-positive XXX cases, maternal XXX was confirmed by karyotype. For the false-positive cases, mean maternal age was significantly higher in monosomy X (P<.001) and lower in XXX (P=.008). CONCLUSION: Noninvasive prenatal testing results for sex chromosome aneuploidy can be confounded by maternal or fetal biological phenomena. When a discordant noninvasive prenatal testing result is encountered, resolution requires additional maternal history, detailed fetal ultrasonography, and determination of fetal and possibly maternal karyotypes.

A multicenter study directly comparing the diagnostic accuracy of gene expression profiling and immunohistochemistry for primary site identification in metastatic tumors.

Metastatic tumors with an uncertain primary site can be a difficult clinical problem. In tens of thousands of patients every year, no confident diagnosis is ever issued, making standard-of-care treatment impossible. Gene expression profiling (GEP) tests currently available to analyze these difficult-to-diagnose tumors have never been directly compared with the diagnostic standard of care, immunochemistry (IHC). This prospectively conducted, blinded, multicenter study compares the diagnostic accuracy of GEP with IHC in identifying the primary site of 157 formalin-fixed paraffin-embedded specimens from metastatic tumors with known primaries, representing the 15 tissues on the GEP test panel. Four pathologists rendered diagnoses by selecting from 84 stains in 2 rounds. GEP was performed using the Pathwork Tissue of Origin Test. Overall, GEP accurately identified 89% of specimens, compared with 83% accuracy using IHC (P=0.013). In the subset of 33 poorly differentiated and undifferentiated carcinomas, GEP accuracy exceeded that of IHC (91% to 71%, P=0.023). In specimens for which pathologists rendered their final diagnosis with a single round of stains, both IHC and GEP exceeded 90% accuracy. However, when the diagnosis required a second round, IHC significantly underperformed GEP (67% to 83%, P<0.001). GEP has been validated as accurate in diagnosing the primary site in metastatic tumors. The Pathwork Tissue of Origin Test used in this study was significantly more accurate than IHC when used to identify the primary site, with the most pronounced superiority observed in specimens that required a second round of stains and in poorly differentiated and undifferentiated metastatic carcinomas.

A gene expression profile test to resolve head & neck squamous versus lung squamous cancers.

BACKGROUND: The differential diagnosis between metastatic head & neck squamous cell carcinomas (HNSCC) and lung squamous cell carcinomas (lung SCC) is often unresolved because the histologic appearance of these two tumor types is similar. We have developed and validated a gene expression profile test (GEP-HN-LS) that distinguishes HNSCC and lung SCC in formalin-fixed, paraffin-embedded (FFPE) specimens using a 2160-gene classification model. METHODS: The test was validated in a blinded study using a pre-specified algorithm and microarray data files for 76 metastatic or poorly-differentiated primary tumors with a known HNSCC or lung SCC diagnosis. RESULTS: The study met the primary Bayesian statistical endpoint for acceptance. Measures of test performance include overall agreement with the known diagnosis of 82.9% (95% CI, 72.5% to 90.6%), an area under the ROC curve (AUC) of 0.91 and a diagnostics odds ratio (DOR) of 23.6. HNSCC (N = 38) gave an agreement with the known diagnosis of 81.6% and lung SCC (N = 38) gave an agreement of 84.2%. Reproducibility in test results between three laboratories had a concordance of 91.7%. CONCLUSION: GEP-HN-LS can aid in resolving the important differential diagnosis between HNSCC and lung SCC tumors. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1753227817890930.

A gene expression profile test for the differential diagnosis of ovarian versus endometrial cancers.

We have developed a gene expression profile test (Pathwork Tissue of Origin Endometrial Test) that distinguishes primary epithelial ovarian and endometrial cancers in formalin-fixed, paraffin-embedded (FFPE) specimens using a 316-gene classification model. The test was validated in a blinded study using a pre-specified algorithm and microarray files for 75 metastatic, poorly differentiated or undifferentiated specimens with a known ovarian or endometrial cancer diagnosis. Measures of test performance include a 94.7% overall agreement with the known diagnosis, an area under the ROC curve (AUC) of 0.997 and a diagnostic odds ratio (DOR) of 406. Ovarian cancers (n=30) gave an agreement of 96.7% with the known diagnosis while endometrial cancers (n=45) gave an agreement of 93.3%. In a precision study, concordance in test results was 100%. Reproducibility in test results between three laboratories was 94.3%. The Tissue of Origin Endometrial Test can aid in resolving important differential diagnostic questions in gynecologic oncology.

Identification of tissue of origin in body fluid specimens using a gene expression microarray assay.

BACKGROUND: Body fluid specimens may be the first and only pathologic specimen for clinical evaluation in metastatic cancer cases. The challenge of identifying the tissue of origin in metastatic cancer has led to the emergence of molecular-based assays, such as the microarray-based Pathwork Tissue of Origin gene expression test. The ability to use body fluid specimens in this test would be valuable in providing diagnoses to cancer patients without clearly identifiable primary sites. In the current study, the authors evaluated the Tissue of Origin Test for use with malignant effusion specimens. METHODS: A total of 27 metastasis-positive body fluid specimens from different body sites, including pleural, ascites, pericardial, and pelvic wash fluids, were obtained from patients with known diagnoses. Nine specimens from nonmalignant body fluids were included as controls. RNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissue and gene expression analysis was performed with the Tissue of Origin Test. RESULTS: Seventeen of 27 metastasis-positive samples were non-necrotic with ≥60% tumor and yielded sufficient RNA. Of these samples, 94.1% (16 of 17) were in agreement with the available diagnosis. Of the 9 negative control samples evaluated, 7 (77.8%) demonstrated microarray expression profiles most similar to lymphoma, which is consistent with the predominance of inflammatory cells in these specimens. CONCLUSIONS: The results of the current study demonstrated that FFPE cell blocks from cytologic body fluid specimens yield adequate diagnostic material for the Pathwork test and can be used in the workup of patients with unknown primary tumors.

Potential clinical utility of gene-expression profiling in identifying tumors of uncertain origin.

AIM: To evaluate the potential impact of a gene-expression-based test on the diagnosis of primary tumors in difficult-to-diagnose cases. MATERIALS & METHODS: The Tissue of Origin Test uses 2000 gene measurements to classify the most likely primary tumor. We categorized 284 consecutive samples by pretest diagnosis, then recategorized the samples using test results to identify cases with changes in diagnosis. RESULTS: A total of 64% of incoming diagnoses were nonspecific. A leading diagnosis for the primary site was provided for remaining cases, indicating an unresolved differential. Overall, the test predicted a change in the most likely primary site, either a change from nonspecific to specific site or a change from one specific primary site to another in 81% of the cases and confirmed the suspected primary site for 15% of cases. CONCLUSION: A new molecular diagnostic has the potential to change both primary site identification and therapy selection for the majority of patients tested.

Beneficial role of the GPR30 agonist G-1 in an animal model of multiple sclerosis.

The beneficial effects of estrogens in multiple sclerosis are thought to be mediated exclusively by the classical nuclear estrogen receptors ERalpha and ERbeta. However, recently many reports revealed that estrogens are able to mediate rapid signals through a G protein-coupled receptor (GPCR), known as GPR30. In the present study, we set out to explore whether effects mediated through this receptor were anti-inflammatory and could account for some of the beneficial effects of estrogen. We demonstrate that GPR30 is expressed in both human and mouse immune cells. Furthermore a GPR30-selective agonist, G-1, previously described by us, inhibits the production of lipopolysaccharide (LPS)-induced cytokines such as TNF-alpha and IL-6 in a dose-dependent manner in human primary macrophages and in a murine macrophage cell line. These effects are likely mediated solely through the estrogen-specific receptor GPR30 since the agonist G-1 displayed an IC(50) far greater than 10 microM on the classical nuclear estrogen receptors as well as a panel of 25 other GPCRs. Finally, we show that the agonist G-1 is able to reduce the severity of disease in both active and passive EAE models of multiple sclerosis in SJL mice and that this effect is concomitant with a G-1-mediated decrease in proinflammatory cytokines, including IFN-gamma and IL-17, in immune cells harvested from these mice. The effect of G-1 appears indirect, as the GPR30 agonist did not directly influence IFN-gamma or IL-17 production by purified T cells. These data indicate that G-1 may represent a novel therapeutic agent for the treatment of chronic autoimmune, inflammatory diseases.

Antiviral and myocyte protective effects of murine interferon-beta and -{alpha}2 in coxsackievirus B3-induced myocarditis and epicarditis in Balb/c mice.

The present study tested the hypothesis that murine (m)IFN-beta or mIFN-alpha(2) can eliminate cardiac viral load and protect cardiomyocytes from injury in animals infected with coxsackievirus B3 (CVB3). CVB3-inoculated male Balb/c mice exhibited signs of illness, including lethargy, progressive weight loss, and death (10% on day 3 and 100% on day 8). Cardiac viral load was high [4,277 +/- 1,009 plaque-forming units and 25 +/- 5 copies CVB3/hypoxanthine guanine phosphoribosyl transferase 1 mRNA] on day 4. The cardiac tissue exhibited severe inflammatory infiltration and myocyte damage with an average myocarditis integrated pathology score of 2.1 +/- 0.2 on day 7. Most of the mice infected with CVB3 also developed epicarditis, and 55% had intraventricular thrombi present. Treatment with mIFN-beta [2.5 to 10 million international units (MIU)/kg] dose-dependently improved the general health status in CVB3-inoculated mice, as evidenced by reduction in weight loss, prevention of death, elimination of cardiac viral load, protection of myocytes from injury, decrease in inflammatory cell infiltration, and attenuation of intraventricular thrombus formation. Treatment with 10 MIU/kg mIFN-alpha(2) resulted in a similar level of efficacy as that induced by 5 MIU/kg mIFN-beta, with the exception that mIFN-alpha(2) did not reduce cardiac CVB3 mRNA. However, mIFN-alpha(2) , but not any dose group of mIFN-beta, significantly attenuated CVB3-induced epicarditis. These data demonstrate antiviral effects for both mIFN-beta and mIFN-alpha(2), which lead to protection of the mice from CVB3-induced myocarditis. However, the potential mechanisms leading to a differential host response for the two isoforms of mIFN remain to be elucidated.

Angiotensin II induces histomorphologic features of unstable plaque in a murine model of accelerated atherosclerosis.

BACKGROUND: We explored the role of angiotensin II in determining the histomorphometric features of plaque stability in apolipoprotein E-deficient mice submitted to ligation of the carotid artery. METHODS: Six-month-old apolipoprotein E-deficient mice underwent ligation of the common left carotid artery and were immediately assigned to receive either angiotensin II (1.4 mg . kg(-1) . d(-1) subcutaneously) or vehicle (phosphate-buffered saline; control) via a subcutaneous osmotic minipump for 4 weeks. RESULTS: Ligated arteries from control animals developed intimal lesions composed of macrophage foam cell plaques, which accumulated adjacent to the internal elastic lamina and were surrounded by a fibromuscular layer. Angiotensin II-treated mice had a greater intimal area (threefold), which was accompanied by a fivefold increase in the foam cell area. Lesions from angiotensin II-treated mice also displayed complex morphology characterized by intralesional neovasculature and hemorrhage. The content of active matrix metalloproteinase 2, mainly colocalized with macrophage foam cells, and the production of the inflammatory mediators monocyte chemoattractant protein 1 and vascular cell adhesion molecule 1 were also increased by angiotensin II treatment. Although angiotensin II induced vessel expansion and lumen loss to a similar extent, only vessel enlargement correlated with intimal area. CONCLUSIONS: Taken together, this study's results support a role of angiotensin II in plaque vulnerability by promoting intraplaque neovascularization/hemorrhage, inflammation, and expansive remodeling.

Identification of a novel prostate tumor target, mindin/RG-1, for antibody-based radiotherapy of prostate cancer.

Gene expression analysis showed that a human mindin homologue, mindin/RG-1, is expressed selectively in prostate tissues and that its expression level is elevated in some prostate tumors. Mindin/RG-1 protein expression is maintained in >80% of prostate cancers metastatic to bone or lymph nodes as well as in locally recurrent tumors in androgen-unresponsive patients. In contrast, mindin/RG-1 expression in other normal tissues is significantly lower than that seen in the prostate. A fully human antibody, 19G9, was generated against mindin/RG-1 protein and was shown to accumulate at high abundance in LNCaP tumor xenografts. Conjugates of this antibody with the chelator CHX-A''-DTPA were generated and radiolabeled with either 111In, 90Y, or 86Y. Small animal positron emission tomography imaging with the 86Y-radiolabeled conjugate showed very specific accumulation of the antibody in LNCaP tumor xenografts with clear tumor delineation apparent at 4 hours. The therapeutic efficacy of [90Y]-CHX-A''-DTPA-19G9 was evaluated in mice bearing LNCaP xenografts. A dose-finding study identified a nontoxic therapeutic dose to be approximately 75 microCi. Significant antitumor effects were seen with a single administration of radiolabeled antibody to animals bearing 200 to 400 mm3 tumors. Inhibition of tumor growth was observed in all treated animals over a 49-day period. At 49 days posttreatment, slow tumor growth recurred but this could be prevented for an additional 40-day period by a second administration of a 75 microCi dose at day 49. We conclude that [90Y]-CHX-A''-DTPA-19G9 is a novel antibody conjugate that has considerable promise for therapy of metastatic prostate cancer in androgen-unresponsive patients.

Inhibition of Rho-kinase by fasudil attenuated angiotensin II-induced cardiac hypertrophy in apolipoprotein E deficient mice.

Recent evidence indicates that the GTPase activated Rho/Rho-kinase pathway contributes angiotensin II-induced cardiac hypertrophy and vascular remodeling. We tested this hypothesis in vivo by determining the effects of fasudil, a Rho-kinase inhibitor, on angiotensin II-induced cardiac hypertrophy, coronary vascular remodeling, and ventricular dysfunction. Six-month-old apolipoprotein E deficient (apoE-KO) mice were subcutaneously infused with angiotensin II (1.44 mg/kg/day) using an osmotic mini-pump. Mice were randomly assigned to either vehicle or fasudil (136 or 213 mg/kg/day in drinking water) group. Infusion of angiotensin II for 4 weeks resulted in cardiac enlargement, myocyte hypertrophy, and myocardial interstitial and coronary artery perivascular fibrosis. These changes were accompanied by reduced aortic flow velocity and acceleration rate. Cardiac gene expression levels of atrial natriuretic peptide (ANP) and collagen type III detected by real-time reverse transcriptase polymerase chain reaction were significantly increased in angiotensin II-infused mice. Treatment with fasudil dose-dependently attenuated angiotensin II-induced cardiac hypertrophy, prevented perivascular fibrosis, blunted the increase in ANP and collagen type III expression, and improved cardiac function, without changing blood pressure. These data are consistent with a role for Rho-kinase activation in angiotensin II-induced cardiac remodeling and vascular wall fibrosis.

Fasudil, a Rho-kinase inhibitor, attenuates angiotensin II-induced abdominal aortic aneurysm in apolipoprotein E-deficient mice by inhibiting apoptosis and proteolysis.

BACKGROUND: Angiotensin II (Ang II) accelerates atherosclerosis and induces abdominal aortic aneurysm (AAA) in an experimental mouse model. Agonism of a G protein-coupled receptor by Ang II activates Rho-kinase and other signaling pathways and results in activation of proteolysis and apoptosis. Enhanced proteolysis and smooth muscle cell apoptosis are important mechanisms associated with AAA. In this study, we tested the hypothesis that fasudil, a Rho-kinase inhibitor, could attenuate Ang II-induced AAA formation by inhibiting vascular wall apoptosis and extracellular matrix proteolysis. METHODS AND RESULTS: Six-month-old apolipoprotein E-deficient mice were infused with Ang II (1.44 mg x kg(-1) x d(-1)) for 1 month. Animals were randomly assigned to treatment with fasudil (136 or 213 mg x kg(-1) x d(-1) in drinking water) or tap water. Ang II infusion induced AAA formation in 75% of the mice, which was accompanied by an increase in proteolysis detected by zymographic analysis and quantified by active matrix metalloproteinase-2 activity, as well as apoptosis detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and quantified by both caspase-3 activity and histone-associated DNA fragmentation. The level of DNA fragmentation in the suprarenal aorta correlated with AAA diameter. Ang II also increased atherosclerotic lesion area and blood pressure. Fasudil treatment resulted in a dose-dependent reduction in both the incidence and severity of AAA. At the higher dose, fasudil decreased AAA by 45% while significantly inhibiting both apoptosis and proteolysis, without affecting atherosclerosis or blood pressure. CONCLUSIONS: These data demonstrate that inhibition of Rho-kinase by fasudil attenuated Ang II-induced AAA through inhibition of both apoptosis and proteolysis pathways.

CCR1 is an early and specific marker of Alzheimer's disease.

Chemokines are a diverse group of small proteins that effect cell signaling by binding to G-protein-coupled, seven-trans-membrane receptors. Our group had found previously that the chemokine receptor CCR1 was present in neurons and dystrophic processes in a small sample of Alzheimer's disease cases. This expanded immunohistochemical study shows that the number of CCR1-positive plaque-like structures in the hippocampus and entorhinal cortex is highly correlated to dementia state as measured by the clinical dementia rating score. CCR1 immunoreactivity is found in dystrophic, neurofilament-positive, synaptophysin-negative neurites that are associated with senile plaques containing amyloid beta peptides of the 1-42 species (Abeta42). CCR1 was not, however, associated with diffuse deposits of Abeta42. There was limited expression of CCR1 in neurofibrillary tangle-bearing neuritic processes. Astrocytes and microglia were typically negative for CCR1. Human brains from age-matched, nondemented individuals rarely displayed either CCR1 or Abeta42 immunoreactivity. Seven other types of dementing neurodegenerative diseases were examined, and all failed to demonstrate CCR1 immunopositivity unless Abeta42-positive plaques were also present. Thus, neuronal CCR1 is not a generalized marker of neurodegeneration. Rather, it appears to be part of the neuroimmune response to Abeta42-positive neuritic plaques.

Urokinase-type plasminogen activator plays a critical role in angiotensin II-induced abdominal aortic aneurysm.

We have previously demonstrated that urokinase-type plasminogen activator (uPA) is highly expressed in the aneurysmal segment of the abdominal aorta (AAA) in apolipoprotein E-deficient (apoE-/-) mice treated with angiotensin II (Ang II). In the present study, we tested the hypothesis that uPA is essential for AAA formation in this model. An osmotic minipump containing Ang II (1.44 mg/kg per day) was implanted subcutaneously into 7- to 11-month-old male mice for 1 month. Ang II induced AAA in 9 (90%) of 10 hyperlipidemic mice deficient in apoE (apoE-/-/uPA+/+ mice) but in only 2 (22%) of 9 mice deficient in both apoE and uPA (apoE-/-/uPA-/- mice) (P<0.05). Although the expansion of the suprarenal aorta was significantly less in apoE-/-/uPA-/- mice than in apoE-/-/uPA+/+ mice, the aortic diameters of the aorta immediately above or below the suprarenal aorta were similar between the 2 groups. Ang II induced AAA in 7 (39%) of 18 strain-matched wild-type C57 black/6J control mice. The incidence was significantly higher in atherosclerotic apoE-deficient (apoE-/-) mice, in which 8 (100%) of 8 mice developed AAA. Only 1 (4%) of 27 uPA-/- mice developed AAA after Ang II treatment. We conclude the following: (1) uPA plays an essential role in Ang II-induced AAA in mice with or without preexisting hyperlipidemia and atherosclerosis; (2) uPA deficiency does not affect the diameter of the nonaneurysmal portion of the aorta; and (3) atherosclerosis and/or hyperlipidemia promotes but is not essential for Ang II-induced AAA formation in this model.