Evaluation of hypermethylated tumor suppressor genes as tumor markers in mouth and throat rinsing fluid,nasopharyngeal swab and peripheral blood of nasopharygeal carcinoma patient

The purpose of our study was to evaluate the frequency of hypermethylated tumor suppressor genes (TSGs) in peripheral blood, mouth and throat (M&T) rinsing fluid and nasopharyngeal (NP) swabs of nasopharyngeal carcinoma (NPC) patients. Six normal NP tissues, 43 M&T rinsing fluid, 37 NP swabs and 43 peripheral blood from healthy non-smokers and non-drinkers without a family history of NPC, and 30 NPC tumors and their matched body fluid were analyzed for the presence of hypermethylated p15, p16, Ras association domain family 1 (RASSF1A), E-cadherin, and death-associated protein kinase (DAPK) by methylation-specific PCR. Sequencing analysis was carried out on selected NPC tumors and body fluid samples. Twenty-nine (97%) tumors displayed methylation in at least 1 of the 5 genes. The methylation frequencies were 80% for p15, 77% for DAPK, 67% for RASSF1A, 53% for E-cadherin and 33% for p16. The frequency range of aberrant methylated genes in the body fluids were NP swabs (17-63%) and M&T rinsing fluid (17-50%). Methylation was found in <20% of peripheral blood for each respective gene. Methylation was, however, detected in 1 M&T rinsing fluid in which the primary tumor showed methylation free for RASSF1A. Five healthy individuals exhibited methylation for DAPK, or RASSF1A, or p15 in their body fluid samples. All body fluid samples of healthy controls showed methylation free for E-cadherin and p16. Epigenetic change is found frequently in NPC and the high detection rate in body fluids suggest its potential application in non-invasive screening of NPC or detection of residual carcinoma after treatment.     

Mechanisms involved in the antiplatelet activity of midazolam in human platelets

BACKGROUND: Midazolam is widely used as a sedative and anesthetic induction agent. The aim of this study was to systematically examine the inhibitory mechanisms of midazolam in platelet aggregation. METHODS: The inhibitory mechanisms of midazolam in platelet aggregation were explored by means of analysis of the platelet glycoprotein IIb-IIIa complex, phosphoinositide breakdown, intracellular Ca+2 mobilization, measurement of membrane fluidity, thromboxane B2 formation, and protein kinase C activity. RESULTS: In this study, midazolam dose-dependently (6-26 microm) inhibited platelet aggregation in human platelets stimulated by agonists. Midazolam also dose-dependently inhibited phosphoinositide breakdown and intracellular Ca+2 mobilization in human platelets stimulated by collagen. Midazolam (6-26 mum) significantly inhibited thromboxane A2 formation stimulated by collagen in human platelets. Moreover, midazolam (15 and 26 mum) dose-dependently decreased the fluorescence of platelet membranes tagged with diphenylhexatriene. Rapid phosphorylation of a platelet protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by collagen (2 microg/ml). This phosphorylation was markedly inhibited by midazolam (26 microm). CONCLUSIONS: These results indicate that the antiplatelet activity of midazolam may be involved in the following pathways: the effects of midazolam may initially be caused by induction of conformational changes in platelet membrane, leading to a change in the activity of phospholipase C, and subsequent inhibition of phosphoinositide breakdown and thromboxane A2 formation, thereby leading to inhibition of both intracellular Ca+2 mobilization and phosphorylation of P47 protein.     

Sinonasal undifferentiated carcinoma and nasopharyngeal-type undifferentiated carcinoma: two clinically, biologically, and histopathologically distinct entities

Sinonasal undifferentiated carcinoma (SNUC) is a rare aggressive neoplasm arising in the nasal cavity and paranasal sinuses. Primary sinonasal nasopharyngeal-type undifferentiated carcinoma (PSNPC) is an even rarer tumor that has not been adequately reported. Both tumors have been reported to be associated with Epstein-Barr virus (EBV). We studied the clinicopathologic features and EBV status of 36 SNUC and 13 PSNPC patients from Taiwan, an EBV endemic area. The median age of SNUC patients was 53 years (range 20-76 years), with a male/female ratio of approximately 2:1. Five patients had histories of previous nasopharyngeal carcinoma treated with irradiation 6-26 years earlier. The most common locations were nasal cavity and ethmoid sinus. Orbital and intracranial invasion and distant metastasis were frequent findings. The median survival was 10 months. All 36 tumors were negative for EBER-1 by in situ hybridization. The median age of PSNPC patients was 58 years (range 36-75 years), with a male/female ratio of approximately 2:1. The most common location is nasal cavity. Eight patients achieved disease-free survival. Eight tumors had the morphology of lymphoepithelioma, whereas significant inflammatory infiltrate was not detected in the other five tumors. All 13 tumors were positive for EBER-1 by in situ hybridization. Because of the difference in the relation with EBV, prognosis, and response to radiotherapy, SNUC and PSNPC should be considered as two entirely different entities. The most important criteria for PSNPC are vesicular nuclei, syncytial pattern, spindle cells, and absence of necrosis.     

Evaluation for synergistic suppression of T cell responses to minor histocompatibility antigens by chloroquine in combination with tacrolimus and a rapamycin derivative,SDZ-RAD

The 4-aminoquinolines, chloroquine and hydroxychloroquine, can suppress chronic graft-versus-host disease (GVHD) following blood and marrow transplantation (BMT) in mice and humans, respectively. We hypothesized that chloroquine in combination with tacrolimus and the rapamycin derivative SDZ-RAD can synergistically suppress T cell responses and antigen-presenting cell (APC) function in vitro. We used the APC-dependent C57BL/6 anti-BALB.B T cell response and APC-independent anti-CD3epsilon antibody-induced response to evaluate the role of synergism between chloroquine and tacrolimus or SDZ-RAD on each component of a T cell response to minor histocompatibility antigens. We found that chloroquine with tacrolimus had a greater synergistic suppression of APC-dependent compared to the APC-independent T cell responses, with a combination index (CIx) for 50% inhibition by mean effect analysis of 0.16 and 0.50, respectively (a lower number indicates greater suppression). By contrast, chloroquine with SDZ-RAD had a similar CIx between the two responsed 0.50 vs0.45) suggesting only T cell suppression. Synergy between chloroquine and SDZ-RAD involved a direct effect on T cell cytokine production, whereas synergism between chloroquine and tacrolimus was due to an effect on both T cells and APCs. We conclude that the renal-sparing 4-aminoquinolines may be used syneristically with immunosuppressive drugs currently used for BMT.     

Interactive exploration of microarray gene expression patterns in a reduced dimensional space

The very high dimensional space of gene expression measurements obtained by DNA microarrays impedes the detection of underlying patterns in gene expression data and the identification of discriminatory genes. In this paper we show the use of projection methods such as principal components analysis (PCA) to obtain a direct link between patterns in the genes and patterns in samples. This feature is useful in the initial interactive pattern exploration of gene expression data and data-driven learning of the nature and types of samples. Using oligonucleotide microarray measurements of 40 samples from different normal human tissues, we show that distinct patterns are obtained when the genes are projected on a two-dimensional plane spanned by the loadings of the two major principal components. These patterns define the particular genes associated with a sample class (i.e., tissue). When used separately from the other genes, these class-specific (i.e., tissue-specific) genes in turn define distinct tissue patterns in the projection space spanned by the scores of the two major principal components. In this study, PCA projection facilitated discriminatory gene selection for different tissues and identified tissue-specific gene expression signatures for liver, skeletal muscle, and brain samples. Furthermore, it allowed the classification of nine new samples belonging to these three types using the linear combination of the expression levels of the tissue-specific genes determined from the first set of samples. The application of the technique to other published data sets is also discussed.     

Identification of mutations in the arginine vasopressin receptor 2 gene causing nephrogenic diabetes insipidus in Chinese patients

Congenital nephrogenic diabetes insipidus (NDI) is, in most instances, a rare X-linked recessive renal disorder (MIM 304800) characterized by the clinical symptoms of polyuria, polydipsia, and dehydration. The X-linked NDI is associated with mutations of the arginine vasopressin receptor type 2 (AVPR2) gene, which results in resistance to the antidiuretic action of arginine vasopressin (AVP) in the renal tubules and collecting ducts. Identification of mutations in the AVPR2 gene can facilitate early diagnosis of NDI, which can prevent serious complications such as growth retardation and mental retardation. We analyzed three unrelated Chinese NDI families and identified three mutations: R106C, F287L, and R337X. In addition, an A/G polymorphism at cDNA nucleotide position 927 (codon 309L) was identified. A functional expression assay of the R106C and F287L mutants in COS-7 cells revealed that both mutants show significant dysfunction and accumulate intracellular cyclic adenosine monophosphate in response to AVP hormone stimulation. These results facilitate the diagnosis of NDI at the molecular level in the Chinese population, and provide insight into the molecular pathology of NDI. Received: July 4, 2001 / Accepted: December 4, 2001     

Unexpected mortality from the use of E. coli L-asparaginase during remission induction therapy for childhood acute lymphoblastic leukemia: a report from the Taiwan Pediatric Oncology Group.

The relative efficacy and toxicity of E. coli L-asparaginase and epidoxorubicin used in remission induction therapy for childhood acute lymphoblastic leukemia (ALL) were assessed in a randomized trial conducted in Taiwan. All patients had standard-risk ALL, defined as a leukocyte count <10 x 10(9)/l and were aged between 1 and 2 or 7 and 10 years, or a leukocyte count <50 x 10(9)/l and were aged between 2 and 7 years, without evidence of a T cell or mature B cell immunophenotype, central nervous system leukemia or expression of two or more myeloid-associated antigens. Ninety-three patients were randomized to receive E. coli L-asparaginase at 10,000 IU/m2 thrice weekly for nine doses and 108 to receive epidoxorubicin at 20 mg/m2 weekly for two doses during remission induction with daily prednisolone, weekly vincristine and, on day 22, a dose of etoposide plus cytarabine. Patients treated with L-asparaginase had a significantly higher rate of fatal infection with or without hemorrhage than did those who received epidoxorubicin during remission induction (six of 93 vs none of 108, P = 0.009), resulting in a lower rate of complete remission in the former group (93.6 vs 99.1%, P = 0.05). In addition, patients treated with L-asparaginase had a higher frequency of hyperglycemia and hypoalbuminemia. The overall rate of event-free survival was lower in patients treated with L-asparaginase than in other patients (P = 0.06); estimated 3-year rates were 72% (95% confidence interval, 55-89%) and 87.2% (78-96%), respectively. We conclude that L-asparaginase (Leunase) given at 10,000 IU/m2 for nine doses was poorly tolerated and resulted in excessive toxicity, both through its effects as a single agent and possibly through potentiation of etoposide.     

Mechanism of action of dietary chemoprotective agents in rat liver: induction of phase I and II drug metabolizing enzymes and aflatoxin B1 metabolism

A range of potential chemoprotective agents, most of them natural dietary constituents, has been examined for ability to modulate both phase I (cytochrome P450 1A1, 1A2, 2B1/2, 2C11, 2E1, 3A, 4A) and phase II drug metabolizing enzymes (glutathione S-transferases, in particular subunits Yc2 and P, aflatoxin B1-aldehyde reductase and quinone reductase) in rat liver. In addition to assays of total enzyme activity and Western blots for individual isozymes, the ability of microsomes to metabolize aflatoxin B1, and of cytosols to conjugate aflatoxin B1 (AFB1)-epoxide to GSH and to produce AFB1-dialcohol, were measured. Induction of gamma-glutamyl transpeptidase activity was examined by histochemistry. Differing patterns of induction were observed, reflecting differences in the control of expression of the individual enzymes studied. Of the compounds examined, butylated hydroxytoluene, ethoxyquin, indole-3-carbinol and phenethyl isothiocyanate were the most potent bifunctional agents (inducing both phase I and II activities). Oltipraz, while only weakly inducing CYP1A2 and 2B1/2, was a potent inducer of phase II enzymes. Caffeic acid, garlic oil, sinigrin and propyl gallate all showed some ability to induce phase II enzymes. 4-Methyl catechol, alpha-tocopherol and red wine decreased certain phase I enzyme activities, while inducing total GST activity. Butylated hydroxytoluene, ethoxyquin, garlic oil and indole-3-carbinol induced gamma glutamyltranspeptidase in periportal hepatocytes. Particularly because of their ability to induce the detoxifying activities of glutathione S-transferase Yc2 and aldehyde reductase, butylated hydroxytoluene, ethoxyquin, indole-3-carbinol, oltipraz, phenethyl isothiocyanate and sinigrin will be effective blocking agents in rodents, if administered prior to AFB1. While these studies indicate the relative contributions of phase I and II metabolism in the overall protective effect in rat, care should be taken that a similar balance is achieved in man, and that relevant enzymes or iso forms are induced.      

Ultrasound detection and quantitation of left to right aortopulmonary shunt flow in a canine model

To test the sensitivity and accuracy of pulsed Doppler ultrasonography to detect and quantitate left to right aortopulmonary shunt flow, an arterial allograft aortopulmonary anastomosis was constructed in nine adult mongrel dogs. Cardiac output and allograft flow were measured as the diameter of the allograft was varied. Piezoelectric crystals attached to the carotid artery and proximal descending aorta were energized with 20 MHz pulsed Doppler signals. Negative Doppler shift and negative Doppler shift/positive Doppler shift were calculated for seven dogs. All dogs exhibited negative Doppler shift in the carotid artery at zero allograft flow; five of the seven dogs exhibited a similar pattern in the descending aorta. Increasing negative Doppler shift was measured in all dogs from both sites as the allograft flow increased. Excellent linear correlation existed between allograft flow and negative Doppler shift and negative Doppler shift/positive Doppler shift for each dog from both sampling sites. However, marked interanimal variation in the slopes of the linear regression lines existed, making the composite linear correlation very poor. Detection of small left to right aortopulmonary shunting and single measurements to quantitate accurately left to right aortopulmonary shunting introduce errors due to intersubject variation. However, these results suggest that serial ultrasound measurements made over a short time can accurately predict changes in left to right aortopulmonary shunting.     

A new monoclonal antibody, TRPM-3, binds specifically to certain rat macrophage populations: immunohistochemical and immunoelectron microscopic analysis

An anti-macrophage monoclonal antibody designated TRPM-3 was produced using thioglycollate-elicited rat peritoneal macrophages as immunogen. By the immunoperoxidase method, TRPM-3 was found to be specific for certain macrophage populations, such as marginal zone macrophages and marginal metallophils of the spleen, sinus macrophages of the lymph nodes, and omentum macrophages. No epithelial cells, peripheral blood monocytes, granulocytes, or lymphocytes had reacted with TRPM-3. Immunoelectron microscopically, reaction to TRPM-3 was clearly demonstrated on the plasma membrane of splenic marginal zone macrophages, lymphatic sinus macrophages, omentum macrophages, and peritoneal macrophages. Accordingly, TRPM-3 was considered to have recognized the particular membrane antigen expressed by restricted macrophage populations.