Diagnosis of tuberculosis.
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Bacteriological Techniques↗
Biomedical subjects
P R Wood
Publications and source records attributed to P R Wood.
Antibody-dependent cell-mediated cytotoxicity to newly excysted juvenile Fasciola hepatica in vitro is mediated by reactive nitrogen intermediates.
Passive intraperitoneal transfer of sera from Fasciola hepatica-infected sheep, cattle or rats can protect naive rats from F. hepatica infection, suggesting a parasite killing mechanism within the peritoneal cavity that is dependent on the presence of parasite-specific antibody. We investigated antibody-dependent cell-mediated cytotoxicity by resident peritoneal lavage cell populations, containing large numbers of monocytes/macrophages, as a potential host resistance mechanism by which juvenile flukes could be killed within the peritoneal cavity of naive rats. Comparative studies were conducted using cell populations containing large numbers of monocytes/macrophages from sheep. The results demonstrate that monocyte/macrophage-rich lavage cell populations from rat and sheep differ substantially in their ability to generate nitric oxide. Only resident rat peritoneal lavage cells were able to mediate antibody-dependent cell-mediated cytotoxicity against newly excysted juvenile liver fluke. The mechanism of cytotoxicity was dependent on, and directly proportional to, the production of nitric oxide and required attachment of effector cells to the newly excysted juvenile liver fluke tegument, which occurred following the addition of sera from F. hepatica-infected animals. This is the first report demonstrating a mechanism of cell-mediated cytotoxicity to newly excysted juvenile liver fluke.
Animals↗
BOVIGAM: an in vitro cellular diagnostic test for bovine tuberculosis.
BOVIGAM which is based on the detection of gamma interferon (IFN- gamma) is a rapid, laboratory assay of a cell mediated immune response that may be used for the detection of tuberculosis (TB) infection in animals. Whole blood is first incubated overnight with bovine PPD, avian PPD or negative control antigens, and IFN- gamma in the supernatant plasma is then measured by EIA. TB infection is indicated by a predominant IFN- gamma response to bovine PPD. Since 1988, BOVIGAM has been extensively trialed on more than 200 000 cattle in Australia, Brazil, Ireland, Northern Ireland, Italy, New Zealand, Romania, Spain and the USA. Sensitivity has varied between 81.8% and 100% for culture-confirmed bovine TB and specificity between 94% and 100%. The IFN- gamma assay detects M. bovis infection earlier than the skin test and in New Zealand is applied to detect skin-test negative cattle with TB, where after slaughter a significant number of IFN- gamma reactors have TB. BOVIGAM is also approved in New Zealand for serial testing skin test positive cattle when non-specificity is suspected. Cattle are tested 7-30 days after a positive caudal fold test. The boosting effect of the skin test on T-cell activity allows blood to be cultured with PPD up to 30 h after collection without effecting accuracy. The BOVIGAM results are not affected by poor nutritional condition and are only mildly and briefly affected by dexamethasone treatment and parturition. IFN- gamma responses of cattle vaccinated with BCG are dose-dependent and short-lived. The BOVIGAM kit is now used routinely in many countries for the detection of M. bovis infected cattle, buffalo and goats.
Animals↗
Characterisation of monoclonal antibodies to ovine interleukin-6 and the development of a sensitive capture ELISA.
A purified recombinant ovine (rOv) interleukin-6 (IL-6) was used to generate specific murine monoclonal antibodies (mAbs) and a polyclonal rabbit antisera to this cytokine. From the 31 initial hybridoma cell lines generated, three stable clones were established which secreted mAbs to rOvIL-6, as judged by a direct enzyme-linked immunosorbent assay (ELISA) and Western blotting. Their specificity was further confirmed by demonstrating that none of the mAbs recognised any of the six other irrelevant recombinant ovine cytokines tested by direct ELISA. All three mAbs displayed cross-reactivity with human and African green monkey IL-6 as demonstrated by direct ELISA and Western blotting. In contrast, the polyclonal antibodies only cross-reacted with bovine IL-6 and not with either of the human or monkey homologues. By combining a mAb with the polyclonal antisera a sensitive, IL-6-specific, capture ELISA was developed that had a sensitivity of 150 pg/ml. This detection system was unequivocally validated by demonstrating that native OvIL-6 could be detected in efferent lymph draining from a stimulated popliteal lymph node. In addition, one of the mAbs was shown to allow the detection of OvIL-6 by intracellular cytokine staining and flow cytometry.
Animals↗
Juvenile Fasciola hepatica are resistant to killing in vitro by free radicals compared with larvae of Schistosoma mansoni.
Free radicals have previously been shown to kill the immature stages of the trematode, Schistosoma mansoni but their effect on newly excysted juvenile (NEJ) flukes of Fasciola hepatica has not been established. Using acetaldehyde and xanthine oxidase to chemically generate reactive oxygen intermediates (ROI), up to 61% of NEJ were killed but only when exposed to high levels of ROI. At low concentrations of acetaldehyde and xanthine oxidase as sources of reactive oxygen intermediates, only 6-29% of NEJ were killed compared with 70-92% of schistosomula. Incubation with lipopolysaccharide (LPS)-stimulated rat peritoneal lavage cells (PLCs) killed only 7-15% of NEJ whereas 78-87% of schistosomula were killed under the same conditions by a mechanism dependent on the production of reactive nitrogen intermediates. Relative to immature and adult parasites, NEJ expressed 2.5-20-fold lower levels of superoxide dismutase and glutathione S-transferase but no catalase activity was detected. Incubation of NEJ with inhibitors of peroxidases and glutathione metabolism increased the mean killing of NEJ by LPS-stimulated rat PLCs to 40-75%. These results demonstrate that, in comparison to schistosomula of S. mansoni, NEJ of F. hepatica are relatively resistant to killing by free radicals and this resistance could, in part, be due to the activity of oxidant scavenger enzymes of NEJ.
Aldehyde Oxidoreductases↗
Trichostrongylus colubriformis extract upregulates TNF-alpha receptor expression and enhances TNF-alpha sensitivity of L929 cells.
Many pathogens have developed strategies to avoid the host's immune system and hence improve their long-term survival. These strategies include antigenic variation, mimicry of host regulatory proteins and production of immunoregulatory molecules. The ruminant gastrointestinal nematode Trichostrongylus colubriformis produces several factors with homology to human immunoregulatory proteins. However, direct immunomodulation by T. colubriformis proteins has not yet been unequivocally demonstrated. Results in the present paper demonstrate that soluble T. colubriformis factors promote proliferation of the TNF-susceptible mouse fibrosarcoma cell line L929, while inhibiting proliferation of all other cell types tested. In addition, T. colubriformis homogenate enhanced the susceptibility of L929 cells to the cytotoxic action of ovine TNF-alpha. Within 1 h of exposure, T. colubriformis factors bind L929 cells in a stable fashion, yet it takes up to 24 h for the cells to become sensitised to TNF-alpha. Interestingly, the increase of both TNF-alpha sensitivity and proliferation of treated L929 cells correlated with an upregulation in expression of TNF-alpha p55 and p75 receptors.
Animals↗
The expression and biologic effects of ovine interleukin-4 on T and B cell proliferation.
Using the reverse-transcriptase polymerase chain reaction (RT-PCR), cDNA encoding ovine (Ov) interleukin-4 (OvIL-4) was generated from mitogen-stimulated peripheral blood mononuclear cells (PBMC). Two identical clones generated from separate RT-PCR reactions differed from a published OvIL-4 sequence, although they had a high degree of identity with the bovine and human homologs. We show by sequence analysis that the OvIL-4 cDNA retained the four alpha-helix structure and disulfide bonds identified in human IL-4 (HuIL-4). Moreover, the cDNA encoding OvIL-4 was expressed in insect cells using the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) as a vector. Supernatants from insect cells infected with the recombinant virus secreted an additional protein with a relative molecular mass of 17,000. This protein was recognized by an anticervine IL-4 monoclonal antibody (mAb) in a Western blot and did not react with any proteins in supernatants from uninfected insect cells or cells infected with the wild-type AcMNPV. Supernatants from insect cells infected with the recombinant virus induced the proliferation of activated B cells in a dose-dependent manner and typically demonstrated 5 x 105 dilution U/ml of activity. However, OvIL-4 had no effect on the proliferation of resting T cells isolated from efferent lymph and actually inhibited the ability of a mitogen to stimulate these resting lymphocytes. In contrast, OvIL-4 induced the proliferation of mitogen-activated lymphoblast, demonstrating the complex role(s) OvIL-4 plays in the regulation of B and T cells.
Adjuvants, Immunologic↗
Ovine interleukin-12: analysis of biologic function and species comparison.
Interleukin-12 (IL-12) is a heterodimeric cytokine produced mainly by phagocytic and antigen-presenting cells (APC). The cDNA encoding the ovine IL-12 (OvIL-12) subunits, p40 and p35, were generated from concanavalin A (ConA)-stimulated peripheral blood mononuclear cells (PBMC). The ovine genes encoded proteins that had the highest amino acid identity to caprine p40 (99% amino acid identity) and p35 (97% amino acid identity) and also displayed a high degree of identity with human p40 (84%) and p35 (79%) homologs. To ensure the equal expression of both subunits, we used the self-cleaving properties of the 2A oligopeptide from foot-and-mouth disease virus (FMDV) to express IL-12 as a single, long open reading frame (ORF) encoding p402Ap35. Using an in vitro transcription/translation system, we demonstrated that this 2A oligopeptide mediated cleavage of the p402Ap35 into p402A and p35, in a manner similar to the processing of the FMDV polypeptide. Moreover, when expressed in COSm6 cells, this self-processing polypeptide encoded a functional heterodimer, which elicited biologic activities associated with IL-12 in other species.
Amino Acid Sequence↗
Assessing quality of life in Mexican-American children with asthma: impact-on-family and functional status.
OBJECTIVE: To assess the internal consistency reliability and construct validity of two questionnaires, the Impact on Family (IOF) and the Functional Status II (R) (FSIIR), in a Mexican-American population of children with asthma. METHODS: We interviewed 115 Hispanic parents of children with asthma and compared the IOF and FSIIR scores and reliability coefficients for the following subgroups: English or Spanish language and high or low educational level. We assessed the construct validity of the IOF Total score and FSIIR Illness score by examining the relationship between these scores and other health status variables. RESULTS: The IOF Total score and FSIIR Illness score demonstrated acceptable construct validity and reliability for language and education subgroups, although several of the IOF subscales had low reliability. CONCLUSIONS: IOF Total score and FSIIR Illness score can be recommended for use by Spanish- and English-speaking Mexican-American respondents.
Adaptation, Psychological↗
Microalbuminuria following anaphylaxis with general anaesthesia.
Microalbuminuria is increasingly recognized as a marker of pathologies that cause acute systemic capillary leak. We report a case of an anaphylactic reaction to general anaesthesia involving cardiac arrest. In this case the urinary excretion of albumin following resuscitation suggests that severe anaphylaxis is another condition for which microalbuminuria is a sensitive monitor.
Adolescent↗
Bm86 antigen induces a protective immune response against Boophilus microplus following DNA and protein vaccination in sheep.
Vaccination of sheep with a plasmid bearing the full length gene for the tick antigen Bm86 either alone or co-administered with plasmid carrying the ovine genes for the cytokines, granulocyte and macrophage colony stimulating factor (GM-CSF) or interleukin (IL)-1beta induced a relatively low level of protection against subsequent tick infestation. This tick damage reached statistical significance only for the groups which were vaccinated with plasmid encoding for Bm86, co-administered with plasmid encoding for ovine GM-CSF. Antibody titres measured against Bm86 were also low in all groups injected with the Bm86 DNA vaccine. Antibody production and anti-tick effect were significantly less than that achieved by two vaccinations with recombinant Bm86 protein. In all cases only a low level of antigen-specific stimulation of peripheral blood lymphocytes was recorded, as measured either by the incorporation of tritiated thymidine or the release of IFN-gamma. Injection of DNA encoding for Bm86, either alone or with co-administered cytokine genes, did however prime for a strong subsequent antibody response following a single injection of recombinant Bm86 protein in adjuvant. Antibody production nevertheless appeared to be slightly less effective than following two vaccinations with recombinant protein. The persistence of antibody following vaccination was the same regardless of the method of primary sensitization. In all cases the half-life of the antibody response was approximately 40-50 days indicating that, in contrast to results reported in mice, DNA vaccination in sheep did not result in sustained antibody production.
Animals↗
Cytokine release by ovine macrophages following infection with Chlamydia psittaci.
Chlamydia psittaci is an obligate intracellular pathogen that causes abortion in both sheep and humans. The disease in sheep (but not humans) is characterized by a long-term persistent phase that appears to be under the control of interferon-gamma. However, nothing is known about cytokine induction that precedes the persistent phase in sheep. Primary alveolar lavage cells recovered from normal adult sheep were used to study cytokine production in the first 72 h of infection with C. psittaci. These cells were phenotypically characteristic of macrophages, being adherent, phagocytic, CD14+ and staining positive for non-specific esterase. In vitro infection of the macrophages with C. psittaci resulted in the release of IL-1beta, IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) as measured by ovine-specific ELISAs. Heat-treated chlamydiae (1 h at 65 degrees C) did not induce the release of IL-1beta, but the release of IL-8 was similar to that induced by untreated organisms. The cells from different sheep varied most notably in their patterns of GM-CSF release in response to heat-treated and untreated organisms.
Animals↗
Antibody and cytokine responses in efferent lymph following vaccination with different adjuvants.
The cannulated efferent lymph node in sheep was used to examine the effect of different adjuvants on the antibody and cytokine responses following sub-cutaneous vaccination with a recombinant Taenia ovis antigen (45 W). Vaccination with Quil A elicited relatively higher levels of IgM than did IFA or Al(OH)3. In general, 45 W specific IgG1 and IgG2 titres were higher and maintained for longer periods of time in lymph from sheep vaccinated with IFA and lower and shorter lived in animals which received the Al(OH)3 based vaccine. Interferon-gamma was present within one day in efferent lymph from all sheep which received the Quil A formulation and in only one of the three sheep that received the IFA formulation. GM-CSF was only detected in lymph from sheep vaccinated with the IFA formulation. IL-8 was present in lymph prior to vaccination and only animals which received the Quil A formulation had increased levels of IL-8 after vaccination. Neither of the inflammatory cytokines IL-1 beta and TNF alpha were detected in efferent lymph from any animals in this study. This paper highlights the potential of the lymphatic cannulation model for investigations of the in vivo action of adjuvants.
Adjuvants, Immunologic↗
The use of recombinant ovine IL-1beta and TNF-alpha as natural adjuvants and their physiological effects in vivo.
In the present study we have investigated the use of recombinant ovine IL-1beta and TNF-alpha both alone and in combination, as natural adjuvants in vaccination trials in sheep. Initial experiments were conducted to investigate the physiological effects of the cytokines in vivo and determine what dose could be administered without adverse pyrogenic effects. Even at the maximum dose tested (100 microg) the only significant physiological effect was a transient increase in body temperature of approximately 2 degrees C in sheep injected with TNF-alpha. Administration of either cytokine had profound effects on the levels of circulating leucocytes for up to 5 days postinjection. The incorporation of either IL-1beta or TNF-alpha in aqueous or Al(OH)3 vaccine formulations enhanced antibody responses to a recombinant antigen from the cestode parasite Taenia ovis. The addition of IL-1beta to aqueous vaccine formulations increased antibody responses 15-20-fold and in Al(OH)3 formulations by three to six fold. TNF-alpha stimulated 1.5 to six-fold and 2.5 to seven-fold increases in antibody levels in aqueous and Al(OH)3-based formulations, respectively, in a dose-dependent manner. The addition of either cytokine to Quil A or IFA vaccines did not enhance the antibody levels elicited. When 10 microg of both IL-1beta and TNF-alpha were incorporated in the aqueous or Al(OH)3 vaccine formulations, increases of 21-fold and 25-fold, respectively, were observed in antibody levels. The adjuvant activity of IL-1beta and TNF-alpha in combination in the Al(OH)3-based vaccine resulted in antibody levels commensurate with those obtained using Quil A or IFA.
Adjuvants, Immunologic↗
Immunosuppressive therapy with microemulsion cyclosporine A shortens the hospitalization of pediatric liver transplant recipients.
Intravenous administration of cyclosporine (Sandimmune) to rapidly and effectively achieve therapeutic serum levels in transplant recipients has been the treatment standard in many transplantation centers. With the development of microemulsion cyclosporine (Neoral), that standard is changing. Neoral has greater bioavailability than the oral form of Sandimmune and, consequently, can be more efficacious and cost-effective. To test this hypothesis, we undertook a retrospective study of Sandimmune and Neoral in the treatment of 66 children who underwent uncomplicated orthotopic liver transplantation in the Texas Medical Center between April 1991 and December 1997. Both forms of cyclosporine were evaluated in terms of in-patient treatment cost, recuperative time in the intensive care unit and duration of hospitalization. Twenty-two patients were treated orally with Neoral, and 44 patients were treated intravenously with Sandimmune for a mean time of 14 d. Once the blood concentration of Sandimmune reached a steady state, as confirmed by daily measurements of the trough level, the patients in the Sandimmune group were converted to oral cyclosporine. None of the 22 patients treated with Neoral required intravenous treatment. The mean time spent in the intensive care unit was 4 d for the Neoral group and 5.5 d for the Sandimmune group. The mean duration of hospitalization from the date of transplantation to discharge was 12 d for the Neoral group and 20 d for the Sandimmune group (p < 0.001). Based on these results, we determined that the overall cost per patient in the Neoral group was $3598 less than that per patient in the Sandimmune group.
Adolescent↗
Expression of ovine interleukin-2 cDNA in Escherichia coli.
The expression plasmids pGEX-2T and pT7-7 were used to express ovine (Ov) IL-2 cDNA in Escherichia coli. The pGEX-2T vector contained glutathione-S-transferase (GST) as the affinity handle and resulted in high level expression of the GST-IL-2 fusion protein. However, only a small proportion of this fusion protein was present in the soluble fraction. The insoluble fraction was extracted with a detergent, sarkosyl, and even though a large amount of fusion product was obtained, it would not bind to glutathione beads efficiently. Thus, only low yields of biologically active rOvIL-2 were obtained. The yields were not significantly improved when other detergents were used for extraction except for a non-ionic detergent, Zwittergent 3-14, where there was a two- to three-fold increase compared with extraction with sarkosyl. An alternative vector, pT7-7 was used with a 6 x histidine tag followed by a thrombin cleavage site at the amino terminus of the mature ovine IL-2 protein to allow affinity purification by Ni-NTA resin. A large proportion of the rOvIL-2 was partitioned to the insoluble fraction. This expression system was more useful than the pGEX-2T as large quantities of biologically active rOvIL-2 of at least 10 mg l-1 were obtained. The presence of the six histidine residues at the amino end of rOvIL-2 did not reduce its biological activity. Both systems yielded rOvIL-2 with a high specific activity of about 1 x 10(7) U mg-1 as measured by the ability to maintain proliferation of ovine ConA lymphoblasts. Recombinant OvIL-2 was active on bovine but not porcine ConA lymphoblasts.
Animals↗
Analysis of ovine IL-1 beta production in vivo and in vitro by enzyme immunoassay and immunohistochemistry.
A monoclonal antibody (mAb) specific for ovine IL-1 beta was produced and, in conjunction with a polyclonal rabbit antiserum, used to develop a sensitive enzyme immunoassay (EIA) for ovine interleukin 1 beta (IL-1 beta). The mAb neutralised the activity of recombinant ovine IL-1 beta (rOvIL-1 beta) and native OvIL-1 in an ovine thymocyte proliferation assay. However, it did not neutralise the biological activity of rOvIL-1 beta in the murine NOB1/CTLL assay. The mAb did not react with rOvIL-1 alpha, IL-2, IL-4, IL-8, tumor necrosis factor-alpha, gamma-interferon or recombinant human IL-1 beta in indirect EIA. Immunohistological staining of activated alveolar macrophages and frozen lymph node sections sections demonstrated that the mAb detected IL-1 beta secreted by ovine macrophages (CD11c-positive). The EIA was highly sensitive, detecting less than 50 pg ml-1 of rOvIL-1 beta and low levels of native IL-1 beta in supernatants from lipopolysaccharide-stimulated macrophages. The EIA did not detect heat-inactivated IL-1 beta.
Alkaline Phosphatase↗
Urea/DTT solubilization of a recombinant Taenia ovis antigen, 45W, expressed as a GST fusion protein results in enhanced protective immune response to the 45W moiety.
Vaccination and challenge infection experiments were conducted in sheep using different forms of a recombinant protein (45W) from the cestode parasite Taenia ovis. 45W was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (45W-GST) and was produced as both soluble protein and insoluble inclusion bodies. Vaccination of animals with either the soluble or inclusion body derived protein resulted in the immune response being predominantly directed to the GST moiety of 45W-GST. Conversely, vaccination with 45W-GST which had been solubilized/treated with urea and dithiothreitol (DTT), elicited enhanced responses to the 45W moiety and significantly reduced responses to GST. Vaccination with all forms of 45W-GST protected sheep against experimental T. ovis infection. However, protection was highly correlated with anti-45W antibody levels and these were significantly higher in animals vaccinated with the urea/DTT treated form of 45W-GST. It is suggested that recombinant proteins expressed either with or without fusion partners may stimulate enhanced immune responses when incorporated in vaccine formulations in a denatured/reduced state.
Animals↗