Rapid adenosine release in the nucleus tractus solitarii during defence response in rats: real-time measurement in vivo

We have measured the release of adenosine and inosine from the dorsal surface of the brainstem and from within the nucleus tractus solitarii (NTS) during the defence response evoked by hypothalamic stimulation in the anaesthetised rat. At the surface of the brainstem, only release of inosine was detected on hypothalamic defence area stimulation. This inosine signal was greatly reduced by addition of the ecto-5'-nucleotidase inhibitor α,β-methylene ADP (200 μM), suggesting that the inosine arose from adenosine that was produced in the extracellular space by the prior release of ATP. By placing a microelectrode biosensor into the NTS under stereotaxic control we have recorded release of adenosine within this nucleus. By contrast to the brainstem surface, a fast increase in adenosine, accompanied only by a much smaller change in inosine levels, was seen following stimulation of the hypothalamic defence area. The release of adenosine following hypothalamic stimulation was mainly confined to a narrow region of the NTS some 500 μm in length around the level of the obex. Interestingly the release of adenosine was depletable: when the defence reaction was evoked at short time intervals, much less adenosine was released on the second stimulus. Our novel techniques have given unprecedented real-time measurement and localisation of adenosine release in vivo and demonstrate that adenosine is released at the right time and in sufficient quantities to contribute to the cardiovascular components of the defence reaction.     

Acute peripheral arterial and graft occlusion: treatment with selective infusion of urokinase and lysyl plasminogen

Thirty-five patients hospitalized for recent angiographically documented arterial occlusion in the legs (27 femoropopliteal arteries and eight grafts) benefited from local fibrinolytic therapy delivered at the site of the occlusion with a 4- or 5-F catheter. This therapy combined a continuous urokinase (UK) infusion of 1,000 U/kg/hour and a lysyl plasminogen (LYS-PLG) infusion of 15 microkatals every 30 minutes. Angiographically confirmed lysis was obtained in 85% of the cases. Only 3% of the patients had major and 6% had minor groin hematomas. Only two patients had concentrations of fibrinogen as low as 100 mg/dl. Intravascular infusion of UK-LYS-PLG is as effective as streptokinase. Its excellent tolerance makes it a good alternative in the treatment of acute ischemia in the lower limbs.     

What some patients can eat during migraine attacks: therapeutic and conceptual implications

Although nausea and vomiting are diagnostic migraine symptoms, most patients can take tablets by mouth and a few say they can eat some food. This study was conducted to determine the proportion who could eat or drink, what was consumable and with what effect. One-hundred-and-nine migraineurs were asked what they could eat or drink at the beginning or height of their attacks; 59 could not take any food by mouth, but 50 could eat during the headache phase of their migraine attacks. Four ate normally, 5 took smaller amounts of their normal dietary intake, and 3 took lighter meals. Dry, carbohydrate foods were consumable by the remaining 38: a few had specific cravings, most stated the precise variety which, when eaten, reduced nausea, headache, other symptoms or length of attacks. Patients should therefore be encouraged to eat what they can tolerate, with their tablets taken as early as possible after the onset of attacks. Simultaneous nausea, tolerance or even craving for specific foods occur in other conditions, particularly high altitude headaches which share other features of migraine attacks. The observations in this paper support the notion that migraine is a central neuronal metabolic disturbance.     

Screening of Twenty-Four South African Combretum and Six Terminalia Species (Combretaceae) for Antioxidant Activities

The dried leaves of Combretum and Terminalia species (Combretaceae) were extracted with acetone, hexane, dichloromethane and methanol. Thin layer chromatography (TLC) plates were developed under saturated conditions and sprayed with 0.2% 2,2-diphenyl-1-picryl hydrazyl (DPPH) in methanol for antioxidant screening. Visualization of separated bands exhibiting antioxidant activities enabled the localization and the subsequent identification of the potential active compounds. The acetone and methanol extracts displayed the presence of antioxidant activity after spraying the chromatogram with DPPH. Hexane and dichloromethane extracts did not have any antioxidant activity. C. hereroense had the highest number of active compounds, followed by C. collinum ssp. taborense, which were 16 and 10, respectively. Acetone extracts of all tested Combretum species had 53 active bands and methanol had 55. All Terminalia species extracted with acetone and methanol had antioxidant activity. T. gazensis and T. mollis methanol extracts had 11 and 14 active compounds respectively in one of the solvent systems used. The qualitative DPPH assay on TLC was successfully used in this study to systematically assess the total antioxidant activity of the Combretum and Terminalia species extracts.     

Expression of class II MHC gene products by macrophages in human uteroplacental tissue.

The expression of class II MHC determinants by fetal and maternal macrophages in human uteroplacental tissues was examined with monoclonal antibodies directed against HLA-DR, DP and DQ antigens. Maternal macrophages in early and full-term pregnancy decidua were HLA-DR positive, and a substantial proportion also expressed DP and DQ antigens. Fetal macrophages in chorionic villous stroma in first-trimester pregnancy were rarely HLA-DR positive, and DQ and DP antigens were never expressed. In term placental tissues, groups of villous stromal macrophages were HLA-DR positive, as were fetal macrophages within amniotic mesenchyme. In contrast, DP and DQ antigens were detected on a small minority of fetal macrophages in term placental tissues. DP and DQ antigens were not detected in the absence of DR antigens.     

Characterization of endometrial T lymphocyte subpopulations in spontaneous early pregnancy loss

T lymphocyte subpopulations were compared in normal first trimester human decidua and in decidua associated with spontaneous abortion. Cryostat sections were labelled using a panel of monoclonal antibodies specific for CD3, CD8, CD4 and for the alphabeta and gammadelta heterodimers of the T cell receptor using an avidin-biotin complex peroxidase method. All the endometrial T cell subsets which have been demonstrated in normal early pregnancy were detected in similar numbers and proportions in spontaneous abortion. The findings suggest that adverse pregnancy outcome is not influenced by altered proportions of T cell subpopulations; nevertheless, the possibility remains that these cells may have an altered antigenic phenotype in spontaneous abortion which could contribute to pregnancy success or failure.      

Germline mutations of the CDKN2 gene in UK melanoma families

Germline mutations in CDKN2 on chromosome 9p21, which codes for the cyclin D kinase inhibitor p16, and more rarely, mutations in the gene coding for CDK4, the protein to which p16 binds, underlie susceptibility in some melanoma families. We have sequenced all exons of CDKN2 and analysed the CDK4 gene for mutations in 27 UK families showing evidence of predisposition to melanoma. Five different germline mutations in CDKN2 were found in six families. Three of the mutations (Met53Ile, Arg24Pro and 23ins24) have been reported previously. We have identified two novel CDKN2 mutations (88delG and Ala118Thr) which are likely to be associated with the development of melanoma, because of their co-segregation with the disease and their likely functional effect on the CDKN2 protein. In binding assays the protein expressed from the previously described mutation, Met53Ile, did not bind to CDK4/CDK6, confirming its role as a causal mutation in the development of melanoma. Ala118Thr appeared to be functional in this assay. Arg24Pro appeared to bind to CDK6, but not to CDK4. No mutations were detected in exon 2 of CDK4, suggesting that causal mutations in this gene are uncommon. The penetrance of these mutant CDKN2 genes is not yet established, nor is the risk of non-melanoma cancer to gene carriers.      

Phenotypic and functional analysis of human CD3- decidual leucocyte clones.

CD3- leucocyte clones were generated from human first-trimester decidualized uterine endometrium in a culture system containing interleukin-2 (IL-2) and phytohaemagglutinin (PHA). All CD3- clones tested by Southern blot analysis had T-cell receptor (TcR) gamma and delta genes in germ-line configuration. Thirty-six CD3- cell clones obtained from eight decidual samples were mostly CD2+CD56+ but, unlike fresh decidual leucocytes, many were also CD16+. Morphological differences were noted between CD16+CD56+ and CD16-CD56+ clones, with the latter cells possessing granules of more variable size. All CD16+ clones expressed strong cytotoxic activity against natural killer (NK) sensitive and NK-resistant cell targets, while CD16- clones had low or negligible activity. Some CD3- clones produced high levels of interferon-gamma, tumour necrosis factor-negligible activity. Some CD3- clones produced high levels of interferon-gamma, tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) upon stimulation, but there was no relationship between specific cytokine production and cell clone phenotype or cytotoxic function. Levels of TGF-beta were generally higher than those produced by decidual CD3+ T-cell clones. Since decidual CD3- CD16- leucocytes have a low proliferative capacity in response to IL-2, and as clones with this phenotype invariably possess low NK cell activity, it is suggested that the NK cell activity of fresh decidual leucocyte populations is mediated largely by the small numbers of CD3- CD16+ cells present.     

Granulated lymphocytes in human endometrium: histochemical and immunohistochemical studies.

Granulated lymphocytes with an unusual antigenic phenotype (CD56+ CD38+ CD2 +/- CD3- CD16-) form a substantial proportion of leukocytes in human endometrial stroma. The purpose of this study was to examine morphological and antigenic heterogeneity in endometrial granulated lymphocytes (eGL) in imprint preparations, paraffin-embedded sections and frozen sections. eGL in decidual imprints showed variations in cell size, nuclear size, shape and chromatin content and the number and size of cytoplasmic granules. eGL were detected in paraffin-embedded sections using phloxine tartrazine, alcian blue and toluidine blue stains. There was no difference in the number of eGL among the three stains but the granules appeared smaller and more regular when stained with toluidine blue. The proportion of stromal cells which were leukocytes increased from 8.2% in proliferative endometrium to 31.7% in early pregnancy decidua. The number of CD56+ and CD38+ cells increased in late secretory endometrium; CD56+ cells formed greater than 75% of the leukocytes in first trimester decidua. The increased number of CD2+ cells in decidua was not comparable with CD56+ and CD38+ cells suggesting that a lower proportion of CD56+ cells in first trimester decidua coexpress CD2, an observation which was supported by double labelling studies. eGL therefore show morphological and antigenic heterogeneity and the study of granulated lymphocytes in pathological endometrium and decidua will require careful phenotypic and morphological analysis of accurately dated samples.     

Kinetics of lymphocyte transformation in mice immunized with viable avirulent forms of Cryptococcus neoformans.

A murine model was developed to study the cell-mediated immune response of mice immunized with one of two live, avirulent forms of Cryptococcus neoformans: a nonencapsulated mutant and a thinly encapsulated pseudohyphal variant. A lymphocyte transformation assay was used to evaluate the cellular response of control and sensitized spleen cells after in vitro incubation with three merthiolate-killed whole-cell antigens of C. neoformans. An antigen-to-spleen cell ratio of 10:1 and 5 days of incubation of antigen-spleen cell mixtures were established as optimal conditions for maximum lymphocyte transformation. Maximum responses occurred from 2 to 3 weeks after the last of eight weekly intraperitoneal inoculations of C. neoformans. This assay provided an accurate, reproducible method of studying cell-mediated immunity to C. neoformans, and applications to the study of cryptococcal pathogenesis are proposed.