Biomedical subjects
R C Burton
Publications and source records attributed to R C Burton.
Monoclonal antibody anti-NC-2 identifies a second receptor on cells mediating natural cytotoxicity in mice.
We have previously reported the identification by the murine monoclonal antibody (mAb) 1C4 of the first leucocyte receptor which is involved in natural cytotoxicity (NC) against WEHI-164; the NC-1.1 receptor. We report herein the identification and characterization of a second leucocyte receptor which is involved in NC, NC-2 (MW 50,000), identified by a rat anti-mouse mAb D9 (immunoglobulin G2a; IgG2a). Flow cytometric analysis showed that NC-2 was expressed on < 6% of splenic leucocytes of different inbred mouse strains and 96% of the cells of a mast-cell line which has high NC activity. In vitro treatment of splenic leucocytes with the D9 mAb blocked effector cell-WEHI-164 target cell conjugation and NC by approximately 50% without affecting natural killing (NK). Western blot analysis of affinity purified NC-2 and NC-1.1 using the D9 and 1C4 mAbs showed specific reactivity of the proteins with D9 and 1C4, respectively. Pretreatment of splenic leucocytes with both mAbs blocked NC 84%, a result which almost doubled that caused by either mAb alone. Flow cytometric screening of 16 different mouse cell lines showed that 19% of the cell lines expressed both receptors, 6% expressed only NC-2, 44% expressed mainly or only NC-1.1 and the remaining cells expressed neither receptor. These data indicate that D9 identifies a xeno-antigen, NC-2, which is expressed on cells mediating NC and not NK, and that it is not the previously described NC-1.1 allo-antigen. We conclude that NC-2 is likely to be one of a number of receptor molecules on cells mediating NC against tumour cells.
Screening for colorectal cancer--the AHTAC report: where do we go from here?
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Phosphorylation of the NC-1.1 receptor and regulation of natural cytotoxicity by protein kinase C and cyclic GMP-dependent protein kinase.
Natural cytotoxicity (NC) against cancer involves receptor-ligand interactions between lymphohemopoietic cells that mediate NC against tumor cells. The only candidate for a receptor on cells mediating NC is NC-1.1, identified using mAb 1C4. In this study we showed that mAb 1C4 blocked NC-1.1+ cell conjugation to WEHI-164 tumor cells, indicating that NC-1.1 is a surface protein required for cell-cell interaction. Affinity-purified NC-1.1 was a 45-kDa monomeric protein. It was a good in vitro substrate for cyclic GMP (cGMP)-dependent protein kinase (PKG) and protein kinase C (PKC) and a relatively poor substrate for cAMP-dependent protein kinase (PKA). Phosphopeptide mapping revealed one phosphopeptide phosphorylated by PKG and PKA, and two additional peptides phosphorylated by PKC. Phosphorylation by PKG or PKA abolished phosphorylation at the PKC sites, while coincubation of NC-1.1 with both PKG and PKC reduced phosphorylation of all sites. NC-1.1 was also a phosphoprotein after immunoprecipitation from intact spleen cells and its phosphorylation was increased after cell stimulation with PKC or PKG activators (phorbol esters or 8-bromo-cGMP). The possible consequences of intracellular signaling were tested in functional assays for NC. Phorbol ester activation of spleen cells increased NC, while 8-bromo-cGMP and 8-bromo-cAMP had little effect. However, coincubation with both phorbol ester and either 8-bromo-cGMP or 8-bromo-cAMP virtually abolished NC without affecting cell conjugation. These results suggest that NC-1.1 is a receptor for a ligand on certain tumor cells and reveal that key intracellular signaling pathways involving PKC, PKG, and PKA interact to effect a coordinated control of NC.
Natural cell-mediated cytotoxicity (NCMC) against NK-sensitive tumours in vitro by murine spleen Ly-6C+ natural T cells.
Ly-6C+ cells constitute 13 +/- 3% of freshly isolated (CBA x C57BL/6)F1 mouse spleen leukocytes. Three distinct populations were identified: CD3 epsilon +NK-1.1- conventional T cells (6%), CD3 epsilon -NK-1.1- granulocytes (5%) and CD3 epsilon +NK-1.1+ T cells (approximately 2%). The CD3 epsilon +NK-1.1+ cells displayed a predominantly large granular leukocyte morphology and were the only Ly-6C+ cell subset identified by MAb 2B6-F2 to spontaneously lyse the NK-sensitive YAC-1 tumour in vitro. On further phenotypic analysis, these cells co-expressed high levels of TCRV beta 8.1/8.2 and CD11b, moderate levels of CD90 and low levels of CD4 or CD8. The removal of CD4+ and CD8+ cells prior to Ly-6C+ cell sorting showed that it was the CD4-CD8- double-negative (DN) CD3 epsilon +NK-1.1+ T-cell subset which was responsible for killing YAC-1. These results indicate that we have identified a DN Ly-6C+ subset of the recently designated NK-1.1+TCR alpha beta low natural T (NT) cells, which are capable of natural cell-mediated cytotoxicity (NCMC) against the NK-sensitive YAC-I tumour in vitro. Additionally, these cells mediated the in vitro killing of 2 further NK-sensitive tumours, murine B16 melanoma and human Jurkat T lymphoma. YAC-1 and Jurkat expressed Fas and were susceptible to anti-Fas MAb or rhuman Fas ligand (rhFasL)-induced lysis. Furthermore, anti-human Fas MAb M3 was shown to block sorted Ly-6C+ splenocyte in vitro killing of Jurkat. In contrast, B16 did not express cell-surface Fas and was resistant to anti-Fas MAb-induced lysis. Taken together, these results show that not only do Ly-6C+ NT cells kill NK-sensitive tumours in vitro but they mediate this activity via multiple cytotoxic mechanisms including Fas.
TNF-alpha is not the sole mediator of WEHI-164 tumour cell killing in natural cytotoxicity.
Using a mAb to NC-1.1, a receptor involved in recognition of tumour targets, the authors have examined the dogma that murine natural cytotoxicity (NC) is exclusively mediated by TNF-alpha. Three different NC-1.1+ spleen cells, WEHI-3BR1 myelomonocytic cells and an uncloned mast cell line-MCL) were reacted with NC-sensitive WEHI-164 targets in vitro, and the induction of TNF-alpha mRNA, surface expression of TNF-alpha, and the appearance of apoptotic bodies in the culture were simultaneously measured. NC-1.1+ spleen cells and WEHI-3BR1 cells showed marked induction of TNF-alpha mRNA within 30 min and this was maintained for up to 18 h. Only transient TNF-alpha mRNA induction was observed in MCL cells at 30 min. Surface TNF-alpha was detected on WEHI-3BR1 cells by 4 h, but was not detected on MCL cells. All three effector cell types mediated NC against WEHI-164 targets within 18 h, but they responded differently to the addition of anti-TNF-alpha mAb: anti-TNF-alpha completely blocked WEHI-3BR1 NC, blocked NC-1.1+ spleen cell NC by approximately 70%, and did not block NC by MCL cells. This indicates that TNF-alpha is induced during NC by WEHI-3BR1 effectors and NC-1.1+ spleen cells, is the sole mediator of NC by WEHI-3BR1, and appears to play no role in NC by MCL cells.
The antitumor effects of levamisole in mice are mediated by NC-1.1+ cells.
Murine natural cytotoxicity, which is a major component of the innate immune response in cancer, is mediated by leukocytes that express the NC-1.1 receptor. Mice depleted of natural cytotoxicity by treatment with an anti-NC-1.1 mAb show enhanced growth of certain transplantable tumors, so agents that enhance natural cytotoxicity by NC-1.1+ cells have the potential to be effective anticancer therapeutic agents. We have examined the immunomodulatory effect of levamisole on natural cytotoxicity mediated by NC-1.1+ cells against the BALB/c WEHI-164 murine fibrosarcoma. Administration of levamisole to BALB/c mice significantly enhanced in vitro splenic natural cytotoxicity against 51Cr-labeled WEHI-164 tumor cells. The effect was most marked 48 h after levamisole treatment, at a dose of 10 mg/kg body weight. This enhancement of natural cytotoxicity by levamisole could be completely abrogated by pretreatment of mice with an anti-NC-1.1 mAb. Treatment of BALB/c mice with 10 mg/kg levamisole significantly reduced the growth of WEHI-164 and this effect was abrogated by pretreatment of mice with anti-NC-1.1, indicating that the antitumor effect of levamisole was mediated, at least in part, via NC-1.1+ cells.
Quality of life after surgery for early breast cancer.
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An IL-3-induced splenic NC-1.1+ mast cell line mediates natural cytotoxicity independent of TNF-alpha.
Long-term culture of mouse spleen cells in IL-3-conditioned medium induced a stable mast cell line. This mast cell line (MCL) which could not be cloned expressed NC-1.1, a receptor on cells which mediate natural cytotoxicity (NC), and CD32/CD16 but no markers of T cells, B cells, macrophages, or NK cells. The MCL cells were large and granular, with abundant cytoplasm and >90% stained with Alcian blue, a mast cell-specific stain. Probing total RNA with cDNA encoding a mast cell-specific proteinase mMCP-5 identified the approximately 1-kb mMCP-5 transcript which was confirmed at the protein level. MCL cells mediated very high NC against WEHI-164 which increased with time in culture and which was blocked by anti-NC-1.1 but not by anti-TNF-alpha. When incubated with WEHI-164 tumor cells, MCL cells showed transient induction of TNF-alpha mRNA, but no detectable surface protein. Thus long-term culture of murine spleen cells in IL-3 induces mast cells which express the NC-1.1 receptor of cells which mediate NC, and utilize a cytotoxic effector mechanism which is not TNF-alpha.
Ly-6C+ natural T (NT) cells mediate immune surveillance against NK-sensitive and NK-resistant transplantable tumours in certain strains of mice.
We have previously shown that anti-Ly-6C monoclonal antibody (MAb) 2B6-F2 identifies a subset of (CBA +/- C57BL/6)F1 splenic NK-1.1+ natural T (NT) cells which kill the NK-sensitive YAC-1 target in vitro. Furthermore, these Ly-6C+ cells are responsible for 40-50% of in vitro YAC-1 killing in all mouse strains tested. In BALB/c and DBA/2 mice, these cells killed not only YAC-1 but also the NK-resistant WEHI-164 M1/16 target via a receptor that recognises a shared determinant on these targets in vitro. In the present study, the anti-tumour role of Ly-6C+ cells against the NK-sensitive B16 melanoma and NK-resistant tumours WEHI-7 T lymphoma and WEHI-164/1C fibrosarcoma was studied in vitro and in vivo. In vitro, B16, WEHI-7 and WEHI-164/1C tumour cell lines were highly sensitive to Ly-6C+ cell killing. In vivo, these same tumours showed significantly increased growth when transplanted s.c. into syngeneic mice treated with 2B6-F2 (0.05 < or = p < 0.0005), and this was most marked in the first 15 days following tumour appearance, when tumours were <15 mm in diameter. Our results show that Ly-6C+ cells play a role in controlling the growth of transplantable NK-sensitive B16 melanoma, and in BALB/c mice, at least, the repertoire of susceptible tumours is extended to include NK-resistant WEHI-7 and WEHI-164/1C. We conclude that Ly-6C+ NT cells play a role in immunosurveillance against NK-sensitive as well as NK-resistant tumours in certain strains of mice.
Has mortality from melanoma stopped rising in Australia? Analysis of trends between 1931 and 1994.
OBJECTIVE: To describe recent trends in mortality from melanoma in Australia. DESIGN: An analysis of trends in age standardised and age and sex specific mortalities by year of death and median year of birth (cohort). SETTING: Australia. SUBJECTS: All deaths from melanoma registered in Australia between 1931 and 1994. RESULTS: Melanoma mortality rose steadily from 1931 to 1985. From 1959 the annual rate of increase was 6.3% in men and 2.9% in women, resulting in mortalities of 4.82 and 2.51 per 100,000 person years in 1985 and 1989, respectively. Mortalities for both sexes seem to have plateaued from June 1985 onwards. In 1990-4 the rate rose by 3.7% in men to 5.00 per 100,000 and in women it fell by 5.2% to 2.38 per 100,000. The non-significant increase after 1985 in mortality in men was restricted to those aged over 70 years of age, whereas the fall in rates in women was mostly in those aged under 55 years. This pattern was generally reflected in the state trends, though with some variation: rates for women in Queensland had peaked in the late 1970s; while rates for men in New South Wales continued to rise in 1990-4, placing them above those for Queensland. Examination of mortalities specific for age, period, and cohort for Australia as a whole showed several salient features. Rates in men rose steeply in cohorts born before about 1930; were stable in cohorts born between 1930 and 1950; and fell in more recent cohorts. Rates in women showed similar changes but about five years earlier. CONCLUSION: Melanoma mortality in Australia peaked in about 1985 and has now plateaued. On the basis of trends in cohorts it can be expected to fall in coming years.
Screening for melanoma by primary health care physicians: a cost-effectiveness analysis.
BACKGROUND AND DESIGN - Australia has the highest rates of skin cancer in the world, and the incidence is estimated to be doubling every 10 years. Despite advances in the early detection and treatment of melanoma about 800 people still die nationally of the disease each year. A possible strategy for further reducing the mortality from melanoma is an organised programme of population screening for unsuspected lesions in asymptomatic people. Arguments against introducing melanoma screening have been based on cost and the lack of reliable data on the efficacy of any screening tests. To date, however, there has been no systematic economic assessment of the cost effectiveness of melanoma screening. The purpose of this research was to determine whether screening may be potentially cost effective and, therefore, warrants further investigation. A computer was used to simulate the effects of a hypothetical melanoma screening programme that was in operation for 20 years, using cohorts of Australians aged 50 at the start of the programme. Based on this simulation, cost-effectiveness estimates of melanoma screening were calculated. RESULTS - Under the standard assumptions used in the model, and setting the sensitivity of the screening test (visual inspection of the skin) at 60%, cost effectiveness ranged from Aust$6853 per life year saved for men if screening was undertaken five yearly to $12 137 if screening was two yearly. For women, it ranged from $11 102 for five yearly screening to $20 877 for two yearly screening. CONCLUSION - The analysis suggests that a melanoma screening programme could be cost effective, particularly if five yearly screening is implemented by family practitioners for men over the age of 50.
Analysis of public education and the implications with regard to nonprogressive thin melanomas.
Public education campaigns to encourage early diagnosis of melanoma have been conducted in a number of countries with predominantly white populations for more than a decade. During the mid to late 1980s the incidence of melanoma has increased sharply above long-term trends in some of these populations. There is evidence that these campaigns can lead to earlier diagnosis of the disease, and thus advancement of the time of diagnosis certainly accounts for some of the recent incidence increases. However, it is possible that earlier diagnosis has also uncovered a preexisting nonfatal (nonmetastasizing) form of melanoma, and that this accounts for a substantial proportion of increases in incidence. Although this should not alter public education for early diagnosis of melanoma, it should give caution to current enthusiasm for population screening for the disease.
Monoclonal antibody 2B6-F2 identifies a subset of murine natural killer cells with receptors for YAC-1 and WEHI-164 targets.
This study reports the generation of rat MoAb 2B6-F2 (IgG2a) that identifies a subpopulation of murine NK cells in the spleen, bone marrow, peripheral blood, lung, and peritoneal leukocytes. By flow cytometry, 2B6-F2 reacted with 10% of naive CBA (immunizing strain) spleen leukocytes of which 3% exhibited small lymphocyte morphology while 7% were large granular leukocytes. This pattern of binding was similarly obtained for BALB/c, C57BL/6, CE, and DBA/2 mice. 2B6-F2 and complement reduced splenic NK activity 40-57% in CBA, C57BL/6, BALB/c, CE, and DBA/2 strains. In CBA, C57BL/6, and CE mice < or = 15% reduction in splenic NC activity was observed while DBA/2 and BALB/c mice displayed 55-73% reduction. Cellular competitive inhibition studies showed that 2B6-F2+ NK cells in BALB/c and DBA/2 mice bind and kill YAC-1 and WEHI-164 target cells via a receptor that recognizes a shared determinant on these targets. Western blot and radioimmunoprecipitation studies indicated that 2B6-F2 identifies a 14-kDa monomeric cell surface molecule.
Phenotype and morphology of murine NC-1.1+ natural cytotoxic cells.
The morphology and phenotype of cells identified by the anti-NC-1.1 mAb, 1C4, were studied in CBA and (CBA x C57BL/6) F1 mice. This mAb blocked splenic natural cytotoxic (NC) cell activity in vitro and in vivo. Single colour flow cytometric analysis showed that 6 +/- 1% of all CBA splenocytes were NC-1.1+, and that granular cells of varying sizes, as defined by their forward and side light scatter, contained the highest proportion of NC-1.1+ cells (29 +/- 7%). When analysed by two colour flow cytometry, the large and the granular NC-1.1+ spleen cells in CBA mice were shown to co-express Thy-1.2 and L3T4 (< or = 33%), Mac-1 (< or = 26%), IgM (< or = 60%) and FcR gamma II and J11d (< or = 100%). In contrast, T cell markers were not co-expressed on the small agranular NC-1.1+ spleen cells. This pattern of marker expression was evident in CBA spleens from 2 days of age. When (CBA x C57BL/6) F1 spleen cells were similarly analysed, 82% of granular NC-1.1+ cells also co-expressed NK-1.1. Single colour analysis of CBA bone marrow, thymus, lymph node and peripheral blood leucocytes revealed that < or = 10% of all cells examined were NC-1.1+ while the most granular cells in these organs were 16-43% NC-1.1+. These results support the 'horizontal lineage' theory that NC cells are cells of different lymphohaemopoietic cell lineages at particular stages of differentiation.
Recent incidence trends imply a nonmetastasizing form of invasive melanoma.
In the mid- to late-1980s white populations in Australia, New Zealand and Scotland showed a sharp increase in melanoma incidence above preceding long-term trends, in some cases as much as doubling in as little as 2 years. Most of this increase was in thin melanomas, (< 1.50 mm thick), and males were more affected than females. Thicker melanomas also generally increased in incidence, particularly in males aged 65 years or older. Examination of Australian Medicare and pathology laboratory data indicated that excision of skin lesions and laboratory diagnosis of pigmented lesions also rose sharply in this period, suggesting that advancement of the time of diagnosis was a likely factor in the increase in melanoma incidence. However the maintenance of new higher incidence levels and the increase in incidence of thicker lesions suggests that advancement of diagnosis cannot explain all of the increase. A real increase in incidence and increasing diagnosis of a preexisting, non-metastasizing form of thin melanoma may also have contributed.
Allogeneic blood transfusion reduces murine pulmonary natural killer (NK) activity and enhances lung metastasis of a syngeneic tumour.
A model was established whereby C57BL/6 (B6) blood injected i.v. into C3H mice 7 days prior to i.v. injection of syngeneic UV-2237 tumour cells significantly increased the number of pulmonary metastases counted 21 days later as compared with levels observed in mice treated with saline, C3H or NZW blood or SRBC. This regimen of B6 allogeneic blood transfusion of C3H mice also significantly depressed splenic and pulmonary NK activity as assayed by lysis of 51Cr YAC-I in vitro and by clearance of 111In YAC-I in vivo respectively. Anti-asialo GMI treatment, which depletes NK activity in vivo, and Poly I:C treatment, which enhances NK activity in vivo, were associated with significantly increased and decreased pulmonary metastasis of UV-2237, respectively, in C3H mice. Depletion of CD4+ and CD8+ T cells had no effect. Cyclophosphamide pretreatment which, among other effects, depletes NK cells, significantly increased pulmonary metastasis of UV-2237 in C3H mice. This was corrected by adoptive transfer of normal C3H spleen cells but not spleen cells from anti-asialo GMI-treated C3H mice or B6-blood-transfused C3H mice. Furthermore, a 1:1 mixture of normal C3H spleen cells with spleen cells from B6-blood-transfused C3H mice also failed to reconstitute the cyclophosphamide-pre-treated C3H mice. We conclude that allogeneic blood transfusion augments pulmonary metastasis of the UV-2237 sarcoma in C3H mice and that the mechanism involves suppression of NK activity.