The role of specific DNA adducts in the induction of micronuclei by N-hydroxy-2-acetylaminofluorene in rat liver in vivo

N-Hydroxy-Z-acetylaminofluorene (N-OH-AAF) was administeredi.p. to male Wistar rats 17 h after partial hepatectomy. Hepatocyteswere analyzed for the presence of micronuclei 7 h, 1, 2, 3 and4 days after injection. N-OH-AAF treatment resulted in a highfrequency of micronucleated hepatocytes at days 3 and 4 (19.5and 19.6 respectively). The frequency of micronucleated hepatocyteswas not increased above control values when hepatocytes wereisolated as early as 7 h, 1 or 2 days after injection. Pretreatmentwith the sulfotransferase inhibitor pentachlorophenol (PCP)45 min before injection of N-OH-AAF almost completely preventedthe formation of micronuclei by N-OH-AAF. Parallel biochemicalstudies indicated that inhibition of sulfation of N-OH-AAF byPCP pretreatment prevented the formation of the N-acetylatedDNA adducts iV-deoxyguanosin-8-yl-AAF and 3-deoxyguanosin-N²-yl-AAFby {small tilde}85%. Total adduct formation to DNA was, however,not lowered because of an increase in the formation of the deacetylatedadduct, N-deoxy-guanosin-8-yl-AAF. The lower frequency of micronucleatedhepatocytes observed in the group pretreated with PCP, did notresult from less proliferative activity in this group as comparedto the group treated with N-OH-AAF alone. Therefore, the decreasein the formation of micronuclei indicates that PCP preventsthe clastogenic damage caused by N-OH-AAF. It is concluded thatthe clastogenicity of N-OH-AAF in rat liver is related to theformation of N-acetylated DNA adducts (i.e. N-deoxyguanosin-8-yl-AAFand/or 3-deoxy-guanosin-N²-yl-AAF) and is not related to theformation of the deacetylated DNA adduct N-deoxyguanosin-8-yl-AF.     

Check samples for laboratory self-assessment in DNA flow cytometry. The National Cancer Institute's Flow Cytometry Network experience

The National Cancer Institute's Flow Cytometry Network (NCI-FCN) is attempting to facilitate the transfer of flow cytometry (FCM) of exfoliated bladder cells from the research laboratory to the clinical laboratory. Demonstrating interinstitutional consistency in FCM analysis of replicate specimens simulating clinical barbotage specimens, fixed to allow easy transportation and storage at room temperature was one specific objective. Simulated barbotage specimens were prepared by mixing cultured aneuploid bladder carcinoma cells with normal or mitogen-stimulated peripheral blood mononuclear cells in different ratios. The samples were fixed in 10% formalin for 30 minutes, stored in buffer, and enucleated with pepsin, pH 1.5, before staining with propidium iodide for FCM DNA analysis. Preservation in ethanol or other common DNA cytochemical reagents was found to be unsatisfactory. In contrast, the formalin-fixed samples showed excellent preservation of quantitative DNA fluorescence and coefficient of variation of histogram peaks for over 2 weeks. Exchange of eight fixed specimens among five network laboratories that analyzed them as "unknowns" showed good overall agreement on histogram data and interpretation, although some noteworthy interlaboratory differences were found. This technique could be used for self-assessment surveys of clinical laboratory performance in DNA FCM of bladder barbotage specimens.     

The unit impulse response procedure for the pharmacokinetic evaluation of drug entry into the central nervous system

The unit impulse response theory has been adapted to characterize the transport profile of drugs into the central nervous system (CNS). From the obtained input function, the cumulative plasma volume (V) cleared by transport into the CNS in time can be calculated. Simulation studies demonstrated that transport governed by passive diffusion resulted in a linear relationship between V and time, while the slope of the line, the blood- brain barrier (BBB) clearance, proved to be an adequate and model independent parameter to characterize drug transport into the CNS. The error in the result of the numerical procedure could be limited to less than 10% of the theoretically predicted value. Superposition of 5 or 10% random noise on simulated data did not result in significant differences between the calculated and theoretically predicted clearance values. Simulations of carrier-mediated transport resulted in nonlinear transport curves; the degree of nonlinearity, and thus the detectability, was dependent on the initial degree of saturation of the system, the rate of desaturation, as caused by drug elimination processes and the noise level on the data. In vivoexperiments in the rat were performed, using atenolol, acetaminophen, and antipyrine as model drugs. Linear transport relationships were obtained for all drugs, indicating that transport was dependent on passive diffusion or a low affinity carrier system. BBB- clearance values were 7±1 l/min for atenolol, 63±7 ul/min for acetaminiphen and 316±25 l/min for antipyrine. These experiments validate the applicability of the presented technique in in vivostudies.     

Primary cerebral neuroblastoma (neurocytoma) in adults

Summary We report a case of a third ventricular neuroblastoma (neurocytoma) in a 66 year old man. A stereotactic needly biopsy was performed to obtain a tissue diagnosis and was followed by total resection. We elected not to give radiation or chemotherapy and to follow the patient closely with serial CT scans. Presently, 48 months postoperatively, the patient is free of tumor by head CT scan and able to live independently. We reviewed the literature of primary cerebral neuroblastomas/neurocytomas occurring in adults (15 years of age) and found 32 cases. Our patient is the oldest of this group with a mean age of 32 ± 14 years (S.D.). The location of the 33 neoplasms was intraventricular in 17 cases (52%) and intraparenchymal in 16 cases. The male to female ratio was 2: 1. Of the 17 patients having a minimal follow-up period of 5 months (mean 51 months), five developed recurrences after 5 to 144 months (mean 50 months) compared to 12 patients without recurrence after a 6- to 72-month follow-up period (mean 52 months). Recurrences occurred statistically significantly more often in parenchymal neuroblastomas/neurocytomas than in intraventricular tumor locations.     

Rectal Absorption Enhancement of Des-Enkephalin-γ-Endorphin (DEγE) by Medium-Chain Glycerides and EDTA in Conscious Rats

The stability of the neuroleptic peptide des-enkephalin--endorphin (DEE; Org 5878) in the rectal lumen and the rectal bioavailability of DEE were investigated in conscious rats. Furthermore, the influence of peptidase inhibition, peptidase saturation, and absorption enhancement on DEE bio-availability were evaluated. Na₂EDTA (0.25%, w/v) prolonged the degradation half-life of DEE in the ligated colon from 33 ± 7 to 93 ± 45 min. Without adjuvant, tritium-labeled DEE was absorbed from the rat rectum to a very low extent (0–4%). After administration of an excess of unlabeled DEE or with Na₂EDTA, comparable results were obtained. The medium-chain glyceride preparation MGK markedly enhanced the rectal DEE bioavailability, up to 8–20%, which was further increased to 10–44% by coadministration of Na₂EDTA. No substantial influence of varying the rectal delivery rate was observed. The results suggest that absorption enhancement and enzyme inhibition both are essential for effective increase of rectal peptide bioavailability.     

Clinical characterization of non-small-cell lung cancer tumors showing neuroendocrine differentiation features

In most cases of small-cell lung carcinomas (SCLC) phenotypic features compatible with a neuroendocrine differentiation status can be identified by monoclonal (MOC) antibody-based immunohistological procedures. Similar features can be recognized only in a minority of non-SCLC tumors. During a period of 30 months, all diagnostic non-SCLC biopsies (141 cases) were prospectively analysed for the presence of markers indicative for neuroendocrine differentiation. In 31% of all cases, such a presence could be noticed. Neuroendocrine differentiation (50% to 100% positive-staining tumor cells) was recognized more frequently in adenocarcinoma when compared to large-cell and squamous-cell carcinoma (chi 2 = 9.31, 2 degrees of freedom, P less than 0.01). To investigate whether the clinical behavior of these "neuroendocrine" non-SCLC cases mimics SCLC, a multivariate analysis for prognostic factors was performed. Among other prognostic factors, biopsies containing more than 50% positive-staining tumor cells with the MOC antibody-1 (MOC-1) were recognized as negative prognostic factors.     

Periaqueductal grey CB1 cannabinoid and metabotropic glutamate subtype 5 receptors modulate changes in rostral ventromedial medulla neuronal activities induced by subcutaneous formalin in the rat

This study was undertaken to analyze the involvement of periaqueductal gray (PAG) cannabinoid or group I metabotropic glutamate receptors in the formalin-induced changes on the rostral ventromedial medulla (RVM) ON- and OFF-cells activities. S.c. injection of formalin into the hind paw produced a transient decrease (4-6 min) followed by a longer increase (25-35 min) in tail flick latencies. Formalin also increased basal activity in RVM ON-cells (42+/-7%) and decreased it in OFF-cells (35+/-4%). Intra-PAG microinjection of (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN 55,212-2) (2 nmol/rat), a cannabinoid receptor agonist, prevented the formalin-induced changes in RVM cell activities. Higher dosages of WIN 55,212-2 (4-8 nmol/rat) increased the tail flick latencies, delayed the tail flick-related onset to ON-cell burst, and decreased the duration of OFF-cell pause. Furthermore, WIN 55,212-2 at a dosage of 8 nmol/rat decreased RVM ON-cell (57+/-7%) and increased OFF-cell ongoing activities (26+/-4%). These effects were prevented by N-piperidino-5-(4-chlorophenyl)-1-(2,4dichlorophenyl)-4-methyl-3-pyrazolecarboxamide SR141716A, (1 pmol/rat), a CB1 cannabinoid receptor antagonist, or by 2-methyl-6-(phenylethynyl)pyridine (MPEP 20 nmol/rat), a selective mGlu5 glutamate receptor antagonist. T7-(hydroxyimino) cyclopropa[b]chromen-1alpha-carboxylate ethyl ester (CPCOOE/50 nmol/rat) and (S)-(+)-alpha-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385, 20 nmol/rat), selective mGlu1 glutamate receptor antagonists, were ineffective in preventing the WIN-induced effects. This study suggests that s.c. injection of formalin modifies RVM neuronal activities and this effect is prevented by PAG cannabinoid receptor stimulation. Moreover, the physiological stimulation of PAG mGlu5, but not mGlu1 glutamate receptors, seems to be required for the cannabinoid-mediated effect.     

Mortality in patients with successful initial response to highly active antiretroviral therapy is still higher than in non-HIV-infected individuals

Mortality in HIV-infected patients has decreased dramatically since the introduction of highly active antiretroviral therapy (HAART). We analyzed progression to death in a population of 3678 antiretroviral treatment-naive patients from the ATHENA national observational cohort from 24 weeks after the start of HAART. Mortality was compared with that in the general population in the Netherlands matched by age and gender. Only log-transformed CD4 cell count (hazard ratio [HR] = 0.50, 95% confidence interval [CI]: 0.40 to 0.61 per unit increase) and plasma viral load (HR = 0.30, 95% CI: 0.15 to 0.60, HIV RNA level <100,000 vs. > or = 100,000 copies/mL) measured at 24 weeks and infection via intravenous drug use (IDU) (HR = 0.16, 95% CI: 0.10 to 0.26, non-IDU vs. IDU) were significantly associated with progression to death. For non-IDU patients with 600 x 10 CD4 cells/L and an HIV RNA level <100,000 copies/mL at 24 weeks, mortality was predicted to be 5.3 (95% CI: 3.5 to 8.4) and 10.4 (95% CI: 6.4 to 17.4) times higher than in the general population for 25-year-old men and women, respectively, and 1.15 (95% CI: 1.08 to 1.25) and 1.29 (95% CI: 1.16 to 1.50) times higher for 65-year-old men and women, respectively. Hence, mortality in HIV-infected patients with a good initial response to HAART is still higher than in the general population.     

抗乙酰胆碱受体单链抗体对致病性抗体结合活性的抑制作用

为探讨单链抗体 (ScFv )对重症肌无力 (MG )的特异性免疫治疗作用 ,从分离自MG患者胸腺的抗乙酰胆碱受体(AchR )抗原结合片段Fab6 37构建单链抗体ScFv6 37。应用放射免疫测定法测定了ScFv6 37与刺激抗原人AchR的特异性结合活性以及与相关抗原大鼠AchR和电鳐AchR的交叉反应 ,应用竞争性ELISA测定抑制致病性抗AchR完整抗体IgG6 37与人AchR结合活性。结果发现ScFv6 37只能与人AchR结合 ,不能与大鼠和电鳐的AchR结合。对IgG6 37与人AchR结合的抑制率为 17 5 %～ 32 9%。表明单链抗体对AchR具有一定的“保护”作用。     

Major histocompatibility complex class I peptide presentation after Salmonella enterica serovar typhimurium infection assessed via stable isotope tagging of the B27-presented peptide repertoire

Reactive arthritis (ReA) induced by infection with several gram-negative bacteria is strongly associated with expression of the major histocompatibility complex class I molecule HLA-B27. It is thought that due to the intracellular lifestyle of ReA-inducing bacteria, bacterial fragments can be presented by HLA-B27. Cytotoxic T cells recognizing such bacterial peptides or other induced host peptides could cross-react with self peptides presented in the joints, giving rise to disease. Studies to analyze the B27 peptide repertoire in relation to infection were severely hampered, as complex peptide profiles obtained from separate infected and noninfected cell preparations had to be compared. For this study, we applied a new approach to examine the effect of Salmonella enterica serovar Typhimurium infection on the B27 peptide repertoire presented by the HLA-B*2704 subtype associated with disease. Firstly, we showed that both host cell and S. enterica serovar Typhimurium proteins can be tagged metabolically with stable-isotope-labeled arginine. We then designed experiments so that either the tagged endogenous or tagged bacterial B*2704-presented peptide repertoires from infected cells could be analyzed by mass spectrometry from single peptide preparations that included uninfected controls. Using this new approach, we found no evidence for significant changes in endogenous B*2704 peptide presentation after infection or for any S. enterica serovar Typhimurium-derived B27-bound peptide. In conclusion, the hypothesis that S. enterica serovar Typhimurium induces changes in B27 peptide presentation could not be supported.