摘要:

SummaryWe describe a simple, non‐radioactive, semi‐automated method for measuring lymphocyteccto‐5′‐nucleotidase activity in isolated cell homogenates. The method which uses inosine 5′‐monophosphate (IMP) as a substrate was optimized and requires a total of 4×106lymphocytes. The reference range obtained (38·7–180·0 nmol/h per mg protein; = 106·6) agrees with other isotopic methods which use14C‐IMP. The simplicity, sensitivity (lower limit 10, upper limit 400 nmol/h per mL) and precision (at 114 nmol/h per mL, interday 4·2%s.d.,n= 20; intraday 5·8%,n= 10) makes this method suitable for routine clinical laboratory use.

ISSN:0004-945X    

DOI:10.1038/icb.1990.11

出版商:Blackwell Publishing Ltd    

年代:2017

数据来源: WILEY

摘要:

SummaryWe measured two ectoenzymes, ecto‐5′‐nucleotidase (NT) and dipeptidyl peptidase IV (DP) in the peripheral blood lymphocytes of various groups of HIV‐infected patients because of the previous implied relationship of these enzymes to immune function.NT expressed as mean nmol/h per mg protein (±s.d.) was significantly depressed in the HIV‐seropositive asymptomatic (42 ± 32;P<0·01) and AIDS groups (14±7;P<0·002) when compared with a healthy HIV‐seronegative male population (83 ± 27). The NT activities in asymptomatic HIV‐seropositive and HIV‐seronegative high risk groups (53 ± 30) were not significantly different from one another but both groups had significantly higher enzyme activities than the AIDS group (P= 0·01 and<0·002, respectively). The seronegative high risk and normal healthy group had similar NT activities.DP activities expressed as mean nmol/h per mg protein (± s.d.) in both seropositive asymptomatic (0·188 ± 0038) and high risk seronegative (0·180 ± 005) groups had higher enzyme activities than the healthy seronegative (0·117 ± 0·015;P= 0·02 and 0·05. respectively) and AIDS group (0·096 ± 0·036;P= 0·002 and 0·02. respectively). The healthy seronegative group had DP activities not significantly different to the AIDS groups. Similarly the high risk seronegative and healthy seropositive group had similar DP activities.These results taken together indicate that measurement of both DP and NT should be evaluated prospectively as a monitor of the clinical progression of HIV infection.

ISSN:0004-945X    

DOI:10.1038/icb.1990.12

出版商:Blackwell Publishing Ltd    

年代:2017

数据来源: WILEY

摘要:

SummaryWe have purified the phosphatidylinositol‐phospholipase C enzyme from the bacteriumBacillus thuringiensis.This enzyme is able to release in soluble form molecules which are anchored lo membranes via a glycan‐phosphatidylinositol group. It exhibits a molecular weight of 33–35 kDa. We raised polyclonal antisera against the molecule and used them in immunoblot as well as radioimmunoassays for enzyme detection. This last technique should facilitate monitoring of chromatographic steps during enzyme purification. We coupled antibodies to Sepharose beads in order to remove the enzyme from incubation media. This reagent also proved to be particularly useful in control experiments designed to ascertain that the observed release of molecules is due to the action of the phosphatidylinositol‐phospholipase C enzyme and not to spontaneous release or to cleavage by nonspecific hydrolases. A search for cross‐reactive molecules in other bacterial strains or mammalian tissues gave negative results. This leads to the conclusion that a great diversity exists between phosphatidylinositol‐phospholipases C, even among different bacterial strains.

ISSN:0004-945X    

DOI:10.1038/icb.1990.13

出版商:Blackwell Publishing Ltd    

年代:2017

数据来源: WILEY

摘要:

SummaryPolymorphonuclear leucocyte (PMN) accumulation is associated with damage to airways epithelial cells in bronchitis. bronchiectasis and some forms of asthma. PMNs release several molecules which may mediate this damage, particularly proteases and oxidants. Using anin vitromodel of intact human amnionic epithelial ells (EC) attached to native basement membrane (BM). we evaluated the capacity of several proteases and oxidants to induce detachment of EC from the BM. Maximum desquamation was observed with collagenase, elastase and trypsin, with minimum effective concentrations required to produce 50% EC‐desquamation (MEC50) for highly purified collagenase, pancreatic elastase, human leucocyte elastase, human leucocyte cathepsin‐G (Cath‐G), trypsin, and kallikrein being 1616 ± 989 U/mL, 32·3± 14·7 U/mL, 85·8±26·7 U/mL, 360±20 U/mL, 340±49 BAEE U/mL and 300±23 U/mL, respectively. Urokinasc (20 U/mL) and plasmin (500 U/mL) produced no desquamation in this system. Relatively high concentrations of oxidants also produced detachment (MEC50. for H2O2and HOCl being 0·59±0·006 mol/L and 0·009 ml/L, respectively and pretreatment of EC membranes with non‐detaching concentrations of H2O2rendered them 10 fold more susceptible to protease‐induced desquamation, suggesting synergism. Reduced glutathione (GSH),N‐acetyl cysteine (NAC), ethylenediamine tetra‐acetic acid (EDTA) and 1,10 phenanthroline ablated collagenase induced EC‐detachment. Elastase induced detachment was sensitive to inhibition by phenyl methyl sulfonyl fluoride (PMSF) and α1‐anti‐proteinase (α1‐AP) and. to a lesser extent by aprotinin; trypsin‐induced detachment was ablated by PMSF. α1‐AP and soybean trypsin inhibitor (SBTI) but not by 1,10 phenanthroline or EDTA. Cath‐G induced detachment was profoundly inhibited by SBTI GSH and NAC. These data demonstrate that human EC can be detached from intact BM by several PMN products, including collagenase, Cath‐G and elastase, and that PMN‐mediated detachment tan be prevented by Cath‐G and collagenase, inhibitors. The data suggest a role for proteases, particularly Cath‐G and collagenase, plus oxidants in synergism with proteases, in mediating PMN‐induced EC detachment.

ISSN:0004-945X    

DOI:10.1038/icb.1990.14

出版商:Blackwell Publishing Ltd    

年代:2017

数据来源: WILEY

摘要:

SummaryThere is an urgent need for new, powerful adjuvants suitable for use with sub‐unit and peptide vaccines in humans. We have measured the humoral immune response in BALB/c mice to vaccine formulations using recombinant HBsAg antigens, and gamma inulin and alum adjuvants. Using Merck, Sharpe&Dohme(MSD) HBsAg at 10 μg/mL, high levels of anti‐HBs were generated and geometric mean S/N ratios of 88, 133 and 107 were obtained for alum absorbed vaccine, gamma inulin, and a mixture of the two adjuvants, respectively. A dilution series produced ED50values of 0·08, 0·15 and 0·22 μg/mL respectively. In a second series of experiments comparing alum and algamulin (a complex of gamma inulin and alum), MSD HBsAg induced anti‐HBs levels of 81 and 52, and ED50values of 0·1 and 0·4 when used in conjunction with alum and algamulin, respectively. SKF HBsAg induced anti‐HBs levels of 126 and 111 with alum and algamulin, and ED50values of 0·11 and 0·075. The class, subclass and level of antibody produced in mice boosted with a second dose of vaccine at 21 days was also examined. Both alum and gamma inulin induced higher levels of total antibody, IgGI and minor IgG subclasses than algamulin, or HBsAg alone. Overall, gamma inulin appears to be an equivalent adjuvant to alum, although their mechanisms of action arc different. Mixtures or complexes of the two adjuvants appear to be less effective in inducing humoral immune responses in mice than either alone.

ISSN:0004-945X    

DOI:10.1038/icb.1990.15

出版商:Blackwell Publishing Ltd    

年代:2017

数据来源: WILEY

摘要:

SummaryImmunization of two macaque monkeys with recombinant vaccinia viruses encoding the en v gene of HIV‐1 (VV‐gp l60) resulted in demonstrable levels of gpl60, gpl20 and gp41‐specific immunoglobulins in both animals. The virus used to immunize one of the monkeys additionally expressed the human IL‐2 gene, which encoded human IL‐2 (VV‐gp l60‐IL‐2). No toxic side‐effects of vaccine‐delivered IL‐2 were observed. Despite marked attenuation of virulence by the coexpressed lymphokine, the levels of vector‐specific antibodies in both animals were similar. Some differences in the HIV‐specific reactivity patterns were detected. Serum reactivity of monkey #A56(VV‐gp 160) was directed against gp41, whereas monkey #B58 (VV‐gpl60‐IL‐2) showed a wider range of recognition, with higher antibody titres against the HIV lysate preparation. Furthermore, this study demonstrates the capacity to boost antibody responses to the vector and coexpressed HIV antigens in primates which are already immune.

ISSN:0004-945X    

DOI:10.1038/icb.1990.16

出版商:Blackwell Publishing Ltd    

年代:2017

数据来源: WILEY

摘要:

SummaryA technique is described for the culture of disaggregated rabbit keratinocytes and the production of confluent sheets of immunologically pure keratinocytes suitable for use in transplantation studies. The benefits of various feeder layers, nutrients and growth hormones were examined by the use of paired controls. The ability of the cultured keratinocyte sheets to be removed from the culture vessel intact and survive as viable autografts was demonstrated, thus establishing a new animal model for the investigation of allograft rejection responses to cultured keratinocytes. The benefits of using the rabbit model for such studies and the advantages of this culture technique over a previously described method of rabbit keratinocyte cell culture are discussed.

ISSN:0004-945X    

DOI:10.1038/icb.1990.17

出版商:Blackwell Publishing Ltd    

年代:2017

数据来源: WILEY

摘要:

SummaryThe production of gamma‐interferon (γ‐IFN) and antigen‐specific immunoglobulin isotypes was monitored in sheep given primary and secondary inocula of protein and polysaccharide antigens with or without adjuvants. In efferent lymph from prefemoral nodes draining the site of inoculation, γ‐IFN levels increased within 24 h after injection of adjuvants Quill A, dextran sulfate (DXS) and 6 days after oil adjuvants in saline. The increased synthesis of γ‐IFN was prolonged by 1–2 days by the presence of adjuvanted antigen, but the major stimulus to γ‐IFN production was provided by the adjuvant. Alhydrogel (AH) did not initiate γ‐IFN synthesis. Following a second inocula of antigen in saline, increased levels of γ‐IFN were detected in efferent lymph after 3–4 h, and persisted for at least 2 days. Following cultivation of leucocytes from immunized sheep with specific antigenin vitro, strong positive correlations between levels ofinterleukin (IL) IL‐2, ‐IFN, proliferative responses and antibody titres were observed. However, after analysis of the antigen‐specific isotypes of immunoglobulin (Ig) there was no correlation between the production of γ‐IFN and particular Ig isotypes. Together with findings that AH did not preferentially induce IgG1 or IgG2, these results suggest that the specific subpopulations of helper T lymphocytes which regulate the production of antibody isotypes by differential secretion of γ‐IFN or IL‐4, are not as clearly defined in sheep as in mice.

ISSN:0004-945X    

DOI:10.1038/icb.1990.18

出版商:Blackwell Publishing Ltd    

年代:2017

数据来源: WILEY

摘要:

SummaryAlkylglycerols, inflammation products of cancerous tissues, are potent macrophage activating agents. A briefin vitrotreatment (30 min) of mouse peritoneal tells with a low concentration (50 ng/mL) of dodecylglycerol (DDG) in 10% foetal calf serum supplemented RPMI‐1640 medium (FCS medium) activates macrophages for Fc‐receptor mediated ingestion activity. A serum factor(s) was shown to be required for the activation of macrophages. When non‐adherent cells were treated with rac‐sn‐l(3)‐dodecylglycerol (DDG) in a serum free‐0·1% egg albumin supplemented RPMI medium (EA medium) for 30 min and cultured in FCS medium for 2 h, the resultant conditioned medium contained a signal factor able to activate macrophages (macrophage activating factor). A conditioned medium prepared with electrophoresed serum α2‐globulin fraction in EA medium markedly enhanced activation of macrophages. Incubation of DDG‐treated non‐adherent cell ghosts in EA medium containing α2‐globulin also produced the macrophage activating signal factor. Therefore, it is concluded that a serum factor in α2‐globulin fraction is processed by pre‐existing functions or enzymes of DDG‐treated non‐adherent cell membrane to yield a macrophage activating signal factor.

ISSN:0004-945X    

DOI:10.1038/icb.1990.19

出版商:Blackwell Publishing Ltd    

年代:2017

数据来源: WILEY

摘要:

Book review in this articleGENETIC ENGINEERING FUNDAMENTALS: AN INTRODUCTION TO PRINCIPLES AND APPLICATIONSBy K. Kammermeyer and V. L. ClarkOPPORTUNISTIC INFECTIONS IN PATIENTS WITH THE ACQUIRED IMMUNODEFICIENCY SYNDROMEEdited by G. S. Leoung and J. MillsAIDS AND OTHER SEXUALLY TRANSMITTED DISEASESEdited by R. Richmond and D. WagfieldACYLOVIR THERAPY FOR HERPES VIRUS INFECTIONSDavid A. Baker.

ISSN:0004-945X    

DOI:10.1038/icb.1990.20

出版商:Blackwell Publishing Ltd    

年代:2017

数据来源: WILEY