Methodologic study of direct preparation for chromosome analysis of human solid brain tumors.

Human malignant glioma grown in athymic nude mice (NHG-1) and three freshly resected human solid gliomas were used in the study of factors influencing the direct preparation (DP) for chromosome analysis of human solid tumors. The results showed that: 1) the length of time after the blood supply was obstructed was a major factor in reducing the success rate of DP, i.e., a 2-hour delay resulted in a significantly lowered metaphase number and after 4 hours almost no metaphases could be seen; 2) preserving tumor cells at 4 degrees C may prolong the time limit to about 4 hours; 3) culture medium (RPMI 1640 and Eagle MEM) and bovine calf serum concentration (0%, 10%, 20%, and 30%) did not influence the success rate significantly; 4) colchicine concentration (0.025 micrograms/mL, 0.05 micrograms/mL, 0.1 micrograms/mL) and time of treatment (30 min, 90 min, or 180 min) mainly affected the quality of chromosomes observed but had little effect on the quantity of metaphases that might be obtained. Based on these results, we had a success rate of more than 80% in 72 xenografts and 22 human brain tumors.     

Analysis of antigen specific T cell helper function in first degree relatives of patients with systemic lupus erythematosus (SLE).

Fourteen families with first degree relatives of patients with systemic lupus erythematosus (SLE) were studied for the ability of their members to respond to the synthetic polypeptide antigen (T,G)-A-L. The family members were also tested for their HLA determinants. All SLE patients tested responded to (T,G)-A-L as measured by the production of (T,G)-A-L specific T cell helper factors by their antigen activated T cells, confirming our previous findings that 100% of SLE donors responded to (T,G)-A-L in contrast to 50% responders in a control population of healthy donors. The general defect in the regulation of immune responses in SLE patients was further indicated by the demonstration that an SLE patient who is a daughter of non-responder parents to (T,G)-A-L, responded to this genetically regulated antigen. In contrast to our observations with SLE patients, the genetic regulation of the ability to respond to (T,G)-A-L was shown not to be impaired in healthy first degree family members of SLE patients and the segregation of the immune response potential in these families was as expected from an inherited dominant trait.     

New Techniques: The use of Fresh-Frozen Tissue in Diagnostic Transmission Electron Microscopy

We have examined the effects of freezing on fresh pathologic human tissue specimens with regard to the fine structural preservation of various organelles that can be of diagnostic importance. Specimens included in this study were frozen either in the cryostat (-20°C) or by quenching in isopentane cooled by liquid nitrogen (-115°C). The results indicate that fresh-frozen tissue that is subsequently fixed and processed for transmission electron microscopy (TEM) provides ultrastructural information that is very nearly equal to that of conventionally processed specimens. Additionally, we performed identical freezing experiments using animal (rat) tissue in order to ensure that the results we obtained using a rather limited number of human specimens were valid. The outcome of these animal experiments thoroughly supports our observations using human pathologic tissue. We believe an appreciation of the fact that fresh-frozen tissue is suitable for TEM examination may become a very valuable resource in the surgical pathology laboratory.     

Potential impact of hormonal male contraception: cross-cultural implications for development of novel preparations

The prospect of a hormonal male contraceptive is no longer distant. Data on the potential impact of this improvement in contraceptive provision, however, is limited, particularly between different cultures. We have therefore carried out a multi-centre study to assess men's attitudes to proposed novel hormonal methods. Questionnaire-based structured interviews were administered to men in Edinburgh, Cape Town, Shanghai and Hong Kong. Approximately 450 men were interviewed in Edinburgh, Shanghai and Hong Kong, and a slightly larger group (n = 493) in Cape Town to give samples (n > 150) of black, coloured and white men. Knowledge of existing male and female methods of contraception was high in all centres and groups. The majority of men welcomed a new hormonal method of contraception, 44-83% stating that they would use a male contraceptive pill. Overall, a pill was more acceptable than an injectable form (most popularly given at 3-6 month intervals); long-acting implants were least so except in Shanghai. Familiarity with comparable female methods appeared to influence acceptability, for both oral and injectable methods. Hong Kong was the only centre where a male method (condom) was currently the most commonly used; men there appeared to rate the convenience of condoms highly while being least likely to think that they provided effective protection against pregnancy compared to other centres, and were least enthusiastic about novel male methods. The acceptability of potential male hormonal methods of contraception was high in some groups but showed wide variability, determining factors including cultural background and current contraceptive usage. These results suggest that the emerging emphasis that men should have greater involvement in family planning will be substantiated when appropriate contraceptive methods become available.     

High-throughput cytochrome P450 (CYP) inhibition screening via cassette probe-dosing strategy. II. Validation of a direct injection/on-line guard cartridge extraction-tandem mass spectrometry method for CYP2D6 inhibition assessment

A highly efficient direct injection/on-line guard cartridge extraction-tandem mass spectrometry (DI/GCE-MS-MS) method has been validated for high-throughput evaluation of cytochrome P450 (CYP) 2D6 inhibition potential using human hepatic microsomes and 96-well microtiter plates. Microsomal incubations were terminated with formic acid, centrifuged, and the resulting supernatants were injected for DI/GCE-MS-MS analysis. Due to the novel use of an extremely short C18 guard cartridge, this method exhibits several advantages, such as no sample preparation, excellent on-line extraction, short run time (2.5 min), and minimized source contamination and performance deterioration. The DI/GCE-MS-MS method demonstrates acceptable accuracy and precision for the quantification of dextrorphan, a marker metabolite of dextromethorphan mediated by CYP2D6, in microsomal incubations. The CYP2D6 inhibition assay has been validated using quinidine as a known selective inhibitor of the isoform. The IC50 value (0.20 microM) measured by the new method is in good agreement with the literature value (0.22 microM).     

Rapid non-genomic inhibitory effects of glucocorticoids on human neutrophil degranulation

Background: Glucocorticoids acting as anti-inflammatory or immunosuppressive drugs have been shown to exert most of their effects genomically. Recent findings suggest that non-genomic activity might be relatively more important in mediating the therapeutic effects of high-dose pulsed glucocorticoid. However, few non-genomic anti-inflammatory effects were reported, much less non-genomic mechanisms.Objective: This study was performed to investigate the nongenomic effects of glucocorticoids on human neutrophil degranulation.Methods: Purified human neutrophils were pretreated with 6 -methylprednisolone or hydrocortisone for 5 min, and then primed with N-formyl-methionyl-leucyl-phenylalanine (fMLP) (10^–6 M) or phorbol myristate acetate (PMA) (50 ng/ml) in the presence of cytochalasin B. The release of two markers of neutrophil granules, lactoferrin and myeloperoxidase, was measured by ELISA and enzymology methods respectively.Results: Both 6 -methylprednisolone (10^–5–10^–4 M) and hydrocortisone (10^–4 M) showed significant inhibitory effects on neutrophil degranulation within 5 min after fMLP administration. For PMA stimulated degranulation, 6 -methylprednisolone (10^–4 M) showed significant inhibitory effects (p < 0.01), while hydrocortisone (10^–4 M) only showed an inhibitory tendency (P > 0.05). Neither RU486 (10^–5 M) nor cycloheximide (10^–4 M) could alter the inhibitory effects of glucocorticoids.Conclusion: Our results demonstrate that megadoses of glucocorticoids exert rapid inhibitory effects on human neutrophil degranulation at the cellular level via a new mechanism that is independent of corticosteroid type II receptor occupation or protein synthesis. We infer that these effects may be very important when glucocorticoids act as anti-inflammatory drugs during pulse therapy.Received 20 May 2004; returned for revision 21 July 2004; accepted by M.J. Parnham 23 September 2004L. Liu and Y. X. Wang contributed equally to this work.