Fetal exposure to alcohol as evidenced by fatty acid ethyl esters in meconium in the absence of maternal drinking history in pregnancy

The detection of fatty acid ethyl esters (FAEE) in neonatal meconium has been proposed as a novel screening method for intrauterine exposure to alcohol. We investigated the potential use of meconium FAEE screening in a high-risk neonatal population in the absence of maternal drinking history. One hundred forty-two meconium samples of neonates suspected of intrauterine illicit substance exposure and referred to the Motherisk Laboratory were analyzed for the existence of drugs by enzyme-linked immunosorbent assay (ELISA) and FAEE by gas chromatography-flame ionization detection (GC-FID). A positive FAEE test was previously defined as a cumulative measurement of 7 individual FAEE > or = 2 nmol/g. Seventy-one percent of the samples tested positive for at least 1 illicit drug, with cannabis being the most prevalent (52.3%). Fourteen percent of all samples tested positive for prenatal alcohol exposure, as evidenced by cumulative meconium FAEE > or = 2 nmol/g. Ethyl oleate, linoleate, palmitate, and arachidonate were detected most often and at the highest levels. At least 3 individual FAEE were detected in 95% of all positive samples, and none could be identified by the use of 1 selected FAEE. Significantly elevated levels of FAEE above the baseline and the presence of multiple FAEE species in meconium are exclusive to neonates who have likely been exposed to excessive amounts of alcohol in utero. Babies born to mothers who are suspected to use illicit drugs in pregnancy are at elevated risk for exposure also to alcohol in utero. Meconium FAEE are emerging biologic markers that can potentially facilitate earlier diagnosis and intervention for less apparent forms of alcohol-related disabilities that cannot be confirmed in the absence of maternal drinking history.     

Naltrexone for heroin dependence treatment in St. Petersburg, Russia

Naltrexone may be more effective for treating opioid (heroin) dependence in Russia than in the U.S. because patients are mostly young and living with their parents, who can control medication compliance. In this pilot study we randomized 52 consenting patients who completed detoxification in St. Petersburg to a double blind, 6-month course of biweekly drug counseling and naltrexone, or counseling and placebo naltrexone. Significant differences in retention and relapse favoring naltrexone were seen beginning at 1 month and continuing throughout the study. At the end of 6 months, 12 of the 27 naltrexone patients (44.4%) remained in treatment and had not relapsed as compared to 4 of 25 placebo patients (16%; p<0.05). Since heroin dependence is the main way HIV is being spread in Russia, naltrexone is likely to improve treatment outcome and help reduce the spread of HIV if it can be made more widely available.     

Integrin chains beta1 and alphav as prognostic factors in human metastatic melanoma

The expression pattern of integrin-type cell adhesion receptors is often changed during malignant transformation. In the present work, we studied the prognostic significance of beta1 and alphav integrin chains for survival of patients with metastatic melanoma. The expression levels of beta1 integrin were also compared with those of Bcl-2, an anti-apoptotic protein, the presence of which is associated with treatment response and survival in melanoma. The expression of beta1 and alphav integrins in 68 melanoma metastases obtained from 55 patients treated with combined chemoimmunotherapy was studied by immunohistochemistry using anti-beta1 and anti-alphav antibodies. The patients were divided into two groups (using a cut-off point of >/= 81%) for beta1 integrin expression levels and into three categories (negative/low, median, high) for alphav integrin expression levels. All tumours were positive for beta1 integrin, and the tumours (n = 6) which had the highest alphav score were also strongly positive for beta1 (94%; P = 0.0055). Patients (n = 43) with 80% or less beta1 integrin-positive tumour cells in their samples had a median disease-free survival (DFS) of 17.0 months, and patients (n = 12) with 81% or more beta1 integrin-positive tumour cells had a DFS of only 5.7 months (P = 0.0001). Patients (n = 32) with low alphav integrin expression levels had shorter DFS (median 12.3 months; P = 0.0146) than patients (n = 20) with median expression levels (median 16.7 months; P = 0.0146). However, three patients who had a very strong alphav expression in their tumours had a median DFS of only 1.8 months (P = 0.0146). Median level expression of beta1 integrin was associated with the presence of Bcl-2 in tumour cells (P = 0.0033). Our results suggest that beta1 and alphav integrin chains are independently expressed in metastatic melanoma and may have an effect on the metastatic potential of melanoma cells.     

Requirement of Gbetagamma and c-Src in D2 dopamine receptor-mediated nuclear factor-kappaB activation

The D2 dopamine receptor (D2R) was examined for its ability to mediate nuclear factor-kappaB (NF-kappaB) activation through G proteins. Stimulation of D2R-transfected HeLa cells with its agonist quinpirole induced the expression of a NF-kappaB luciferase reporter and formation of NF-kappaB-DNA complex. This response was blocked by pertussis toxin, and by the Gbetagamma scavengers transducin and beta-adrenergic receptor kinase 1 carboxyl-terminal fragment. Unlike Gi-coupled chemoattractant receptors, D2R activated NF-kappaB without an increase in phospholipase C-beta activity, and the response was only slightly affected by the phosphoinositide 3-kinase inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). In contrast, treatment with genistein and 4-amino-1-tert-butyl-3-(p-methylphenyl)pyrazolo[3,4-d] pyrimidine abolished the induced NF-kappaB activation, suggesting involvement of protein tyrosine kinases. Activation of D2R led to phosphorylation of c-Src at Tyr-418, and expression of a kinase-deficient c-Src inhibited D2R-mediated NF-kappaB activation. The D2R-mediated NF-kappaB activation was not dependent on epidermal growth factor (EGF) receptor transactivation since 4-(3'-chloroanilino)-6,7-dimethoxyquinazoline (AG1478), an EGF receptor-selective tyrphostin used at 1 microM, blocked EGF-induced NF-kappaB activation but not the quinpirole-induced response. In addition, the D2R-mediated NF-kappaB activation was enhanced by over-expression of beta-arrestin 1. These results suggest that D2R-mediated NF-kappaB activation requires Gbetagamma and c-Src, and possibly involves beta-arrestin 1.     

Discovery of distinct protein profiles specific for lung tumors and pre-malignant lung lesions by SELDI mass spectrometry

Objectives: Early lung cancer detection and treatment remain a challenge. The efficacy of surface-enhanced laser desorption/ionization (SELDI) technology in lung cancer detection, has not been defined. This study identifies specific protein peak patterns in malignant lung tumors, and in pre-malignant airways epithelium showing neoplastic transformation. Methods: Lung tumor specimens taken from patients participating in a lung cancer screening study (H. Lee Moffitt Cancer Center, Tampa, FL) were laser capture microdissected to obtain pure cell populations from frozen sections of normal lung, atypical adenomatous hyperplasia (AAH) and malignant tumors. SELDI mass spectrometry was used to generate protein profiles in each epithelial cell type. Results: SELDI mass spectroscopy is highly reproducible in detecting lung tumor-specific protein profiles. Three peaks at 17–23 kDa mass range from tumor cells showed markedly increased compared with normal cells. The peak at 17 250 Da was not detected in any of the normal cells. This peak appeared to be present at low levels in the atypical cell samples. Conclusions: This study demonstrates the feasibility of detecting “malignant” protein signatures from lung tumor and pre-malignant pulmonary epithelium using SELDI mass spectrometry. Although additional study is necessary to validate these patterns as unique diagnostic tools, these “malignant” protein signatures lend themselves to identification of populations at high-risk for lung cancer and for monitoring response to lung cancer chemopreventive agents.     

Radiation enhances the anti-tumor effects of vaccinia-p53 gene therapy in glioma

The overall goal of this study was to analyze the effect and mechanism of radiation in combination with vaccinia viruses (VV) carrying the p53 gene against glioma. Comparison of two alternative treatments of cultured C6 (p53(+)) and 9L (p53(-)) rat glioma cells showed significantly reduced survival for both cell lines, especially 9L, when radiation was applied prior to virus versus radiation alone. High p53 protein expression mediated by VV-TK-p53 was measured in infected cells. Single modality treatment of C6 cells with psoralen and UV (PUV)-inactivated VV-TK-p53 (PUV-VV-TK-53) or radiation significantly decreased survival compared with PUV-inactivated L-15 (PUV-L-15) control virus. However, no difference was observed between radiation and combination treatments of C6 cells. In contrast, radiation followed by PUV-VV-TK-53 resulted in dramatic reduction of 9L cell viability, compared to single modality treatment. Flow cytometry analysis of Annexin-V-stained 9L cells showed that radiation and PUV-VV-TK-53 caused a significant decrease in live cells (17.2%) as compared to other treatments and control (61.6-98.3%). Apoptosis was observed in 37.2% of cells, while the range was 0.7-7.8% in other treatment groups; maximal p53 level was measured on day 7 post-infection. In athymic mice bearing C6 tumors, VV-TK-53 plus radiation in both single and multiple therapies resulted in significantly smaller tumors by day 30 compared to the agents given only once. Immunohistochemical analysis of tumor sections demonstrated p53 protein expression over 20 days after VV-TK-53 treatment. Analysis of blood and spleen cells of mice given multiple combination treatments showed significant splenomegaly, leukocytosis, and increased DNA synthesis and response to mitogen. Multiple combination treatments were also associated with significantly elevated natural killer and B cells in the spleen. There were no overt toxicities, although depression in red blood cell and thrombocyte parameters was noted. Collectively, the data demonstrate that radiation significantly improves the efficacy of VV-mediated tumor suppressor p53 therapy and may be a promising strategy for glioma treatment. Furthermore, the results support the conclusion that the mechanisms underlying the enhanced anti-tumor effect of combination treatment include apoptosis/necrosis and upregulation of innate immune defenses.     

A novel cholinesterase and brain-selective monoamine oxidase inhibitor for the treatment of dementia comorbid with depression and Parkinson's disease

Degeneration of cholinergic cortical neurons is one of the main reasons for the cognitive deficit in dementia of the Alzheimer type (AD) and in dementia with Lewy bodies (DLB). Many subjects with AD and DLB have extrapyramidal dysfunction and depression resulting from degeneration of dopaminergic, noradrenergic and serotoninergic neurons. We prepared a novel drug, TV-3326 (N-propargyl-3R-aminoindan-5yl)-ethyl methylcarbamate), with both cholinesterase (ChE) and monoamine oxidase (MAO) inhibitory activity, as potential treatment of AD and DLB. TV-3326 inhibits brain acetyl and butyrylcholinesterase (BuChE) in rats after oral doses of 10-100 mg/kg. After chronic but not acute treatment, it inhibits MAO-A and -B in the brain by more than 70% but has almost no effect on these enzymes in the small intestine in rats and rabbits. The brain selectivity results in minimal potentiation of the pressor response to oral tyramine. TV-3326 acts like other antidepressants in the forced swim test in rats, indicating a potential for antidepressant activity. Chronic treatment of mice with TV-3326 (26 mg/kg) prevents the destruction of nigrostriatal neurons by the neurotoxin MPTP (N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). In addition to ChE and MAO inhibition, the propargylamine moiety of TV-3326 confers neuroprotective activity against cytotoxicity induced by ischemia and peroxynitrite in cultured neuronal cells that results from prevention of the fall in mitochondrial membrane potential and antiapoptotic activity. These unique multiple actions of TV-3326 make it a potentially useful drug for the treatment of dementia with Parkinsonian-like symptoms and depression.     

Unique resistance of I/LnJ mice to a retrovirus is due to sustained interferon gamma-dependent production of virus-neutralizing antibodies

Selection of immune escape variants impairs the ability of the immune system to sustain an efficient antiviral response and to control retroviral infections. Like other retroviruses, mouse mammary tumor virus (MMTV) is not efficiently eliminated by the immune system of susceptible mice. In contrast, MMTV-infected I/LnJ mice are capable of producing IgG2a virus-neutralizing antibodies, sustain this response throughout their life, and secrete antibody-coated virions into the milk, thereby preventing infection of their progeny. Antibodies were produced in response to several MMTV variants and were cross-reactive to them. Resistance to MMTV infection was recessive and was dependent on interferon (IFN)-gamma production, because I/LnJ mice with targeted deletion of the INF-gamma gene failed to produce any virus-neutralizing antibodies. These findings reveal a novel mechanism of resistance to retroviral infection that is based on a robust and sustained IFN-gamma-dependent humoral immune response.     

Chain-Breaking Antioxidant Capability of Some Beverages as Determined by the Clark Electrode Technique

The protective effects of red wine, tea, and coffee on cancer, atheroclerosis, and other diseases are attributed to the antioxidant activity (AOA) of polyphenols, which are abundant in those beverages. We present a rational procedure for determining the total chain-breaking AOA of beverages with the use of the Clark electrode technique. The procedure is based on the steady monitoring of oxygen consumption accompanying the chain peroxidation of methyl linoleate in Triton X-100 aqueous micelles induced by 2,2'-azobis(2-amidinopropan) dihydrochloride as a source of active free radicals. AOA was characterized by the number of kinetic chains (expressed in concentration units) that could be terminated by a beverage. The procedure was applied to determine the AOA of nine red wines and single samples of green and black teas, white wine, beer, and soluble coffee. The addition of any of the studied beverages to the testing system resulted in pronounced retardation of methyl linoleate oxidation. The period of increase in the oxidation rate with time caused by antioxidant consumption was preceded by a period when the oxidation rate was visibly decreased with time (increase in inhibition). The release of polyphenols from their complexes with other components of beverages was suggested as the most probable mechanism of this event, which was observed for the first time in this study.