A novel near-infrared nanomaterial with high quantum efficiency and its applications in real time in vivo imaging.

Fluorescence imaging signal is severely limited by the quantum efficiency and emission wavelength. To overcome these challenges, novel NIR-emitting K5NdLi2F10 nanoparticles under NIR excitation was introduced as fluorescence imaging probe for the first time. The photostability of K5NdLi2F10 nanoparticles in the water, phosphate buffer saline, fetal bovine serum and living mice was investigated. The fluorescence signal was detected with depths of 3.5 and 2.0 cm in phantom and pork tissue, respectively. Fluorescence spectrum with a significant signal-to-background ratio of 10:1 was captured in living mice. Moreover, clear NIR images were virtualized for the living mice after intravenous injection. The imaging ability of nanoparticles in tumor-beard mice were evaluated, the enrichment of K5NdLi2F10 nanoparticles in tumor site due to the enhanced permeability and retention effect was confirmed. The systematic studies of toxicity, bio-distribution and in-vivo dynamic imaging suggest that these materials give high biocompatibility and low toxicity. These NIR-emitting nanoparticles with high quantum efficiency, high penetration and low toxicity might facilitate tumor identification in deep tissues more sensitively.

An hepatitis B virus surface antigen specific single chain of variable fragment derived from a natural immune antigen binding fragment phage display library is specifically internalized by HepG2.2.15 cells.

Hepatitis B virus surface antigen (HBsAg), a specific antigen on the membrane of hepatitis B virus (HBV)-infected cells, provides a perfect target for therapeutic drugs. In order to mediate successful targeted delivery of these therapies, it is essential to have antibodies that recognize HBsAg with high specificity and affinity. In this report, we constructed a natural immune antigen binding fragments (Fab) antibody phage display library against HBsAg and after three rounds of panning, five Fab fragments with significant HBsAg binding ability were selected and analysed. DNA sequencing revealed that all the light chains had the same sequence, while all the Fd genes exhibited different sequences. For further application, all of the Fab antibodies were reconstructed into single chain antibodies (scFvs) and expressed in Escherichia coli BL21 cells. Indirect enzyme-linked immunosorbent assay analysis demonstrated that all five scFvs maintained a high affinity for HBsAg and could bind HBsAg on the membrane of HBV-infected cells. Indirect fluorescent staining analysis revealed that one of the scFvs (scFv15) could be internalized into HBsAg-positive HepG2.2.15 cells through clathrin-mediated endocytosis pathway. The internalizing scFv15 antibody would have great potential for the targeted delivery of therapeutics to HBV-infected cells.

Selective proapoptotic activity of a secreted recombinant antibody/AIF fusion protein in carcinomas overexpressing HER2.

Apoptosis-inducing factor (AIF) represents a caspase-independent apoptotic pathway in the cell, and a mitochondrial localization sequence-truncated AIF (AIFDelta1-120) can be relocated from the cytoplasm to the nucleus and exhibit a constitutive proapoptotic activity. Here, we generated a chimeric immuno-AIF protein, which comprised an HER2 antibody, a Pseudomonas exotoxin translocation domain and AIFDelta1-120. Human Jurkat cells transfected with the immuno-AIF gene could express and secrete the chimeric protein, which selectively recognized HER2-overexpressing tumor cells and was endocytosed. Subsequent cleavage of truncated AIF from immuno-AIF and its release from the internalized vesicles resulted in apoptosis of tumor cells. Intramuscular injection of the immuno-AIF gene caused significant suppression of tumors and substantially prolonged mice survival in an HER2-overexpressing xenograft tumor model. Our study demonstrates the feasibility of the immuno-AIF gene as a novel approach to treating cancers that overexpress HER2.

Correlation of p53 mutations with resistance to platinum-based chemotherapy and shortened survival in ovarian cancer.

PURPOSE: The p53 tumor suppressor gene plays a central role in cell cycle regulation and induction of apoptosis. We analyzed p53 alterations and their impact on response to chemotherapy and clinical outcome in ovarian cancer patients. EXPERIMENTAL DESIGN: One hundred seventy-eight ovarian carcinomas, snap frozen and stored at -80 degrees C, were analyzed for mutations of the p53 gene (exons 2-11) by single-strand conformation polymorphism and DNA sequencing and for p53 overexpression by immunohistochemistry (monoclonal antibody DO7). RESULTS: p53 mutations were found in 56% (99 of 178) of the tumors, and 62% of these were located in evolutionary highly conserved domains of the gene. Time to progression and overall survival were significantly shortened in patients with p53 mutations compared with wild-type p53 (P = 0.029 and P = 0.014) and patients with mutations in highly conserved domains as opposed to nonconserved domains or wild-type p53 (P = 0.010 and P = 0.007). p53 protein overexpression (>10% positively stained nuclei) was found in 62% (110 of 178). Time to progression and overall survival were shorter in cases with p53 overexpression (cutpoint, 10%: P = 0.071 and P = 0.056) but only marginally significant. Resistance to adjuvant cisplatin or carboplatin chemotherapy was significantly more frequent in patients with p53 overexpression (P = 0.001) or p53 missense mutations (P = 0.008) than patients with normal p53. CONCLUSIONS: p53 alterations correlate significantly with resistance to platinum-based chemotherapy, early relapse, and shortened overall survival in ovarian cancer patients in univariate analysis. In multivariable analysis though, p53 was not an independent prognostic factor.

Neurocutaneous melanosis with epilepsy: report of one case.

Neurocutaneous melanosis is a rare congenital syndrome characterized by the association of large or multiple congenital melanocytic nevi and benign or malignant melanotic tumors in the central nervous system. Patients with neurocutaneous melanosis usually have neurological symptoms early in life that progress rapidly due to the development of increased intracranial pressure or malignant melanoma. We report a 2-month-old female infant with multiple congenital melanocytic nevi and frequent seizure attacks. Magnetic resonance imaging of the brain demonstrated several regions compatible with melanotic deposits. During follow-up for one year, she had normal development and was seizure-free under the treatment of phenobarbital and valproic acid. We suggest that infants with large or multiple congenital melanocytic nevi should receive regular clinical check-up and brain imaging to exclude the possibility of central nervous system lesions.

Conditioned nutritional deficiencies in the cardiomyopathic hamster heart.

BACKGROUND: Evidence indicates that nutritional factors may be important in the maintenance of myocyte structure and energetics. The failing myocardium has been reported to exhibit a depletion of several nutrients that are important for the maintenance of intracellular calcium homeostasis and cellular energetics, and levels of oxidative stress. This nutrient depletion may contribute to the progressive deterioration in myocardial structure and function observed in heart failure. OBJECTIVE: To examine the extent to which advanced cardiomyopathy results in a depletion of nutrients and/or metabolites and antioxidants, and whether supplementation with these nutrients may influence cellular structure or function. SUBJECTS AND METHODS: Cardiomyopathic hamsters were randomly placed to one of the three following diet groups: chow; control gelled diet; or a supplemented gelled diet that provided taurine, carnitine, coenzyme Q10, selenium, vitamins E and C, creatine, thiamine and L-cysteine. After approximately three months of supplementation, one group of hamsters underwent functional testing using a modified Langendorff technique with biopsy samples taken for electron microscopy. Myocardial nutrient concentrations were determined in a second group of diseased and nondiseased hamsters of the same age. RESULTS: Cardiomyopathy resulted in a depletion of vitamin E, creatine, carnitine, taurine and coenzyme Q10. Supplementation resulted in improved cardiac ultrastructure, function and contractility compared with nonsupplemented hamsters. CONCLUSIONS: These studies suggest that heart failure results in 'condition-related nutrient deficiencies' that, once corrected, can significantly impact on heart function and structure.

Alternative and aberrant messenger RNA splicing of the mdm2 oncogene in invasive breast cancer.

mdm2 is part of a complex mechanism that regulates the expression of p53 as well as the function of Rb, p19ARF, and other genes. In humans, mdm2 dysregulation is associated with gene amplification. This study was undertaken to characterize altered mdm2 expression in a cohort of 38 invasive breast cancers and 9 normal breast specimens. Reverse-transcription PCR with primers spanning the entire open reading frame of the mdm2 gene in breast tissue RNA samples generated PCR products of full-length mdm2 (1526 bp) as well as smaller products (653, 281, 254, and 219 bp). Sequence analysis demonstrated that the 653-bp product was an alternatively spliced product (defined as splicing at the exon/intron boundary consensus sites), whereas the 281, 254, and 219 bp mdm2 products were aberrantly spliced products (splicing at sites not considered to be exon/intron boundary sites). Reverse-transcription-PCR with normal breast tissue RNA samples yielded only the 1526-bp product in five samples and the 1526-bp product and the 653-bp product in four samples. The 653-bp alternatively spliced product was expressed in 21% of breast cancers, and the smaller, aberrantly spliced mRNA products (281 bp, 254 bp, and/or 219 bp) were expressed in 16% of breast cancers. The protein products predicted by the alternatively spliced mRNAs and the aberrantly spliced mRNAs lacked either the entire binding domain for p53 or the majority of the binding domain for p53. Immunohistochemical analysis of HER2/neu (c-erbB2), estrogen receptor, progesterone receptor, epidermal growth factor receptor, and p53 protein was performed. p53 sequence alterations were identified by mismatch detection and confirmed by p53 oligonucleotide microarray technology. An association was demonstrated between the expression of aberrantly and/or alternatively spliced mdm2 mRNAs and a lack of progesterone receptor. An association was also demonstrated between mdm2 aberrantly and/or alternatively expression products and the presence of p53 tumor suppressor gene mutations. mdm2 is transcribed from two different promoters: one, p53-dependent, and the other, p53-independent. The 5' untranslated region of the transcripts was evaluated to determine the promoter usage in each breast cancer specimen. No correlation was observed between mdm2 splice products and promoter usage. The presence of aberrant expression products of mdm2 in breast cancer specimens was correlated with a shortened overall patient survival. These observations suggest that mdm2 expression is altered in invasive breast cancer and is associated with more aggressive disease.

Comparison of TP53 mutations identified by oligonucleotide microarray and conventional DNA sequence analysis.

As the rate of gene discovery accelerates, more efficient methods are needed to analyze genes in human tissues. To assess the efficiency, sensitivity, and specificity of different methods, alterations of TP53 were independently evaluated in 108 ovarian tumors by conventional DNA sequence analysis and oligonucleotide microarray (p53 GeneChip). All mutations identified by oligonucleotide microarray and all disagreements with conventional gel-based DNA sequence analysis were confirmed by re-analysis with manual and automated dideoxy DNA sequencing. A total of 77 ovarian cancers were identified as having TP53 mutations by one of the two approaches, 71 by microarray and 63 by gel-based DNA sequence analysis. The same mutation was identified in 57 ovarian cancers, and the same wild type TP53 sequence was observed in 31 ovarian cancers by both methods, for a concordance rate of 81%. Among the mutation analyses discordant by these methods for TP53 sequence were 14 cases identified as mutated by microarray but not by conventional DNA sequence analysis and 6 cases identified as mutated by conventional DNA sequence analysis but not by microarray. Overall, the oligonucleotide microarray demonstrated a 94% accuracy rate, a 92% sensitivity, and an 100% specificity. Conventional DNA sequence analysis demonstrated an 87% accuracy rate, 82% sensitivity, and a 100% specificity. Patients with TP53 mutations had significantly shorter overall survival than those with no mutation (P = 0.02). Women with mutations in loop2, loop3, or the loop-sheet-helix domain had shorter survival than women with other mutations or women with no mutations (P = 0.01). Although further refinement would be helpful to improve the detection of certain types of TPS3 alterations, oligonucleotide microarrays were shown to be a powerful and effective tool for TP53 mutation detection.

The tyrosine kinase p56lck is essential in coxsackievirus B3-mediated heart disease.

Infections are thought to be important in the pathogenesis of many heart diseases. Coxsackievirus B3 (CVB3) has been linked to chronic dilated cardiomyopathy, a common cause of progressive heart disease, heart failure and sudden death. We show here that the sarcoma (Src) family kinase Lck (p56lck) is required for efficient CVB3 replication in T-cell lines and for viral replication and persistence in vivo. Whereas infection of wild-type mice with human pathogenic CVB3 caused acute and very severe myocarditis, meningitis, hepatitis, pancreatitis and dilated cardiomyopathy, mice lacking the p56lck gene were completely protected from CVB3-induced acute pathogenicity and chronic heart disease. These data identify a previously unknown function of Src family kinases and indicate that p56lck is the essential host factor that controls the replication and pathogenicity of CVB3.

Effects of estrogen replacement on infarct size, cardiac remodeling, and the endothelin system after myocardial infarction in ovariectomized rats.

BACKGROUND: Estrogen may increase the long-term survival of women who have suffered from a myocardial infarction (MI). We examined the acute and chronic influence of estrogen on MI in the rat left coronary artery ligation model. METHODS AND RESULTS: Female Sprague-Dawley rats (10 to 12 weeks, n=93), divided into 3 groups (rats with intact ovaries, ovariectomized rats administered 17beta-estradiol [17beta-E(2)] replacement, and ovariectomized rats administered placebo 2 weeks before MI), were randomized to left coronary artery ligation (n=66) or sham-operated (n=27) groups. Ten to 11 weeks after MI, rats were randomly assigned to either (1) assessment of left ventricular (LV) function and morphometric analysis or (2) measurement of cardiopulmonary mRNA expression of preproendothelin-1 and endothelin A and B receptors. Acutely, estrogen was associated with a trend toward increased mortality. Infarct size was increased in the 17beta-E(2) group compared with the placebo group (42+/-2% versus 26+/-3%, respectively; P:=0.01). Chronically, wall tension was normalized through a reduction in LV cavity size with estrogen treatment (419+/-41 mm Hg/mm for 17beta-E(2) versus 946+/-300 mm Hg/mm for placebo, P:=0.039). In the LV, there was a 2.5-fold increase in endothelin B mRNA expression after MI in placebo-treated rats (P:=0.004 versus sham-operated rats) that was prevented in the 17beta-E(2) group (P:=NS versus sham-operated rats). CONCLUSIONS: These results suggest that estrogen is detrimental at the time of MI or early post-MI period, resulting in an increased size of infarct or infarct expansion, but chronically, it can normalize wall tension and inhibit LV dilatation, which may in turn lead to increased long-term survival. Regulation of the endothelin system, particularly the expression of the endothelin B receptor, may contribute to these estrogenic effects.

Susceptibility to myocarditis is dependent on the response of alphabeta T lymphocytes to coxsackieviral infection.

Viral myocarditis is an important cause of heart failure and dilated cardiomyopathy. T lymphocytes are implicated in myocardial damage in murine models of coxsackievirus B3 (CVB3) myocarditis. We used knockout mice lacking CD4 (CD4(-/-)), CD8 (CD8(-/-)), both coreceptors (CD4(-/-)CD8(-/-)), or the T-cell receptor beta chain (TCRbeta(-/-)) to address the contribution of T-cell subpopulations to host susceptibility to CVB3 myocarditis. Severity of disease was magnified in CD8(-/-) mice but attenuated in CD4(-/-) mice, consistent with a pathogenic role for CD4(+) lymphocytes. Elimination of both CD4 and CD8 molecules from T lymphocytes by genetic knockout better protected mice from myocarditis, demonstrating that both CD4(+) and CD8(+) T cells contribute to host susceptibility. The same benefit occurred in TCRbeta(-/-) mice, with prolonged survival and minimal myocardial disease observed after CVB3 infection. Elevated interferon-gamma and decreased tumor necrosis factor-alpha expression are associated with attenuated myocardial damage in CD4(-/-)CD8(-/-) mice. These results show that the presence of TCRalphabeta(+) T cells enhances host susceptibility to myocarditis. The severity of myocardial damage and associated mortality are dependent on the predominant T-cell type available to respond to CVB3 infection. One mechanism by which CD4(+) and CD8(+) T-cell subsets influence the pathogenesis of myocarditis may involve specific cytokine expression patterns.

Tissue expression and immunolocalization of tumor necrosis factor-alpha in postinfarction dysfunctional myocardium.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) is markedly elevated in advanced heart failure. It is not known whether tissue TNF-alpha is elevated in the common setting of myocardial infarction leading to heart failure and what the source of TNF-alpha is. To determine this, we studied the expression and protein localization of TNF-alpha and its 2 main receptors (TNF-R1/R2) in a rat model of large infarction. METHODS AND RESULTS: Male rats were randomized to proximal left anterior descending ligation. The animals were killed on days 1, 3, 10, and 35 after ligation to examine gene expression and protein production of TNF-alpha and TNF-R1/R2 from the infarct, peri-infarct, and contralateral zones of infarcted heart. There was increased TNF-alpha mRNA production throughout the myocardium at day 1, and detectable expression persisted to day 35 after myocardial infarction. The expression of this cytokine is not confined strictly to the infarct or peri-infarct zones but is expressed by cardiac myocytes within the myocardium in the contralateral normal zone. Changes in gene expression are mirrored initially by augmented protein production within the myocytes. Levels of TNF-alpha protein in the infarct and peri-infarct zones rose early to 8- to 10-fold above normal levels and rose to 4- to 5-fold in the contralateral zone. Finally, expression of the TNF-R1 mRNA transcripts was upregulated at days 3 and 10 after ligation in the infarct and peri-infarct zones, suggesting that the signal transduction pathways necessary for TNF-alpha in the heart remain intact as TNF-alpha biosynthesis increases. CONCLUSIONS: TNF-alpha is present early in a model of large myocardial infarction and is sustained into the later stage within the myocardium. Expression of this cytokine is not only confined strictly to the infarct or peri-infarct zone but is expressed by cardiac myocytes within the myocardium contralateral to the infarct. Therefore TNF-alpha production forms a part of an important intrinsic myocardial stress response system to injury.

p53 mutations and expression in ovarian cancers: correlation with overall survival.

The p53 gene is altered in approximately 50% of all human malignancies. p53 overexpression, identified by immunohistochemistry, and p53 mutations, identified by single-strand conformational polymorphism (SSCP) and DNA sequencing, have been described in ovarian cancers. p53 overexpression has been correlated with poor outcome for women with ovarian cancer in some studies. With only limited data, the assumption has been made that p53 overexpression corresponds to p53 mutations. The purpose of this investigation was to assess p53 alterations in ovarian cancer to determine if p53 overexpression corresponds with mutations in the p53 gene, and to assess whether either predicts clinical outcome in ovarian carcinoma. Frozen ovarian carcinoma tumor specimens from 105 patients were analyzed by immunohistochemical staining for p53 expression. SSCP was used to screen for mutations and DNA sequencing was used to confirm the specific mutation in exons 2 to 11, encompassing the entire p53 open reading frame. Those ovarian carcinomas identified as wild-type p53 by SSCP were subjected to automated DNA sequence analysis of the entire open reading frame. Relative to DNA sequence analysis, the sensitivity of SSCP was 85% and the specificity was 98%. Immunohistochemical staining demonstrated that 72 of the 105 (69%) cases had positive immunostaining. SSCP and DNA sequencing identified and confirmed mutations in 60 of the 105 carcinomas (57%). Although there was a statistically significant association between p53 immunostaining and p53 mutations (p = 0.0002), false-negative and -positive results were identified. Tumor grade (p = 0.03), stage (p = 0.08), and overall survival (p = 0.15) were moderately associated with positive p53 immunostaining. Patients with p53 mutations and overexpression had shorter overall patient survival (p = 0.02). The findings demonstrated that, individually, p53 mutations and p53 overexpression were each related to shorter patient survival, but the strongest predictor of outcome was a combination of both mutations and overexpression. Comparisons of overall survival for women with mutations in loop 2, loop 3, and the loop-sheet-helix domains together showed a statistically significant difference in survival compared to survival of women whose ovarian cancers had other mutations (p = 0.046).

Cardiac function and cytotoxic aldehyde production in a murine model of chronic iron-overload.

OBJECTIVES: To determine the relationship between the total chronic dose of iron administered, ex-vivo cardiac function and the concentrations of cytotoxic aldehydes in heart tissue of a murine model. METHODS: In the first experiment, 34 male B6D2F1 mice were randomized to receive intraperitoneal injections of 5, 10 or 20 mg of iron dextran for three weeks, or a placebo control. The mice were subsequently randomized to undergo ex-vivo assessment of cardiac function. In the second experiment, free radical generation, quantified by the presence of 20 separate cytotoxic aldehydes, was assessed in heart tissue of 40 mice that were randomized to receive chronic treatment with various concentrations of iron dextran (100 mg to 300 mg total chronic dose administered), placebo treatment with saline, or no treatment at all (baseline). RESULTS: Iron-loaded groups displayed dose-dependent depressions of heart rate, systolic pressure, developed pressure, coronary pressure, -dP/dt and +dP/dt, and increases in diastolic pressure. Monotonic dose-dependent increases in total heart aldehydes were observed in the iron-treated groups (r-0.97, p < 0.0001), whereas no significant differences were observed between baseline or time-placebo control groups. CONCLUSIONS: While no single mechanism is likely to account for the complex pathophysiology of iron-induced heart failure, our findings show that chronic iron-loading in a murine model results in dose-dependent alterations to cardiac function; and results in free radical mediated damage to the heart, as measured by excess concentrations of cytotoxic aldehyde-derived peroxidation products. This is the first description of the effects of excess iron on cardiac function assessed by an ex-vivo Langendorff technique in a murine model of chronic iron-overload.

A serine elastase inhibitor reduces inflammation and fibrosis and preserves cardiac function after experimentally-induced murine myocarditis.

In viral myocarditis, inflammation and destruction of cardiac myocytes leads to fibrosis, causing progressive impairment in cardiac function. Here we show the etiologic importance of serine elastase activity in the pathophysiology of acute viral myocarditis and the therapeutic efficacy of an elastase inhibitor. In DBA/2 mice inoculated with the encephalomyocarditis virus, a more than 150% increase in myocardial serine elastase activity is observed. This is suppressed by a selective serine elastase inhibitor, ZD0892, which is biologically effective after oral administration. Mice treated with this compound had little evidence of microvascular constriction and obstruction associated with myocarditis-induced ischemia reperfusion injury, much less inflammation and necrosis, only mild fibrosis and myocardial collagen deposition, and normal ventricular function, compared with the infected nontreated group.

Chronic endothelin-1 blockade preserves myocardial contractility in dilated cardiomyopathy.

Endothelin-1 (ET-1) is known to have positive inotropic effects in isolated cardiac muscle strips. ET-1 levels are elevated in congestive heart failure (CHF). We investigated the effects of ET-1 on contractility and cardiac relaxation (lusitropy) of the intact healthy murine heart and myocarditic/cardiomyopathic heart by chronic oral treatment with a mixed ETA/ETB blocker SB217242. Chronic ET-1 blockade of normal hearts resulted in depression of contractility and lusitropy of the normal heart but preservation and enhancement of contractility and lusitropy in myocarditic animals, in which ET-1 cardiac content is elevated. This suggests that ET-1 is important in the basal contractility and relaxation of the normal heart but that its chronic elevation in CHF causes impairment of cardiac systolic and diastolic performance.

Genomic semi-automated cycle sequencing as a sensitive screening technique for p53 mutations in frozen tumor samples.

Single-strand conformational polymorphism (SSCP) is the most widely used method for p53 gene mutation screening. Nucleotide sequence analysis is considered more sensitive for detection of mutations. We established a genomic semi-automated cycle sequencing protocol suitable for p53 gene mutation screening. The technique was applied to 44 SSCP-negative frozen ovarian cancer samples: Eleven mutations (11/44, 25%) were found, 6 point missense mutations, 3 silent point mutations, 1 nonsense mutation and 1 single-base deletion. Heterozygous mutations were readily detectable. Genomic semi-automated cycle sequencing is a sensitive, time-effective screening method requiring only small amounts of tumor tissue.