Biomedical subjects
Publications and source records attributed to M Ferguson.
Monoclonal escape variants isolated from the Sabin strain of type 2 poliovirus were examined. Mutations were identified in regions known to be antigenic in other serotypes, although the extent to which they were linked by common antibodies varied. One complex site involved regions of VP2 and VP3 which were not linked in early studies, but have recently been shown by others to be linked in type 1.
The poliovirus/human immunodeficiency virus (HIV) chimera S1/env/3 presents the sequence DRPEGIEEEGGERDRDRS, a known glycoprotein gp41 neutralizing domain (residues 735 to 752) of HIV IIIB in an antigenic site of the Sabin type 1 strain of poliovirus. Of 10 monoclonal antibodies raised against the sequence as presented in S1/env/3, eight were shown to neutralize HIV IIIB in vitro whereas all 10 neutralized S1/env/3, suggesting that the presentation of the sequence is comparable between HIV and the poliovirus/HIV chimera. The monoclonal antibodies were characterized by the selection of escape mutants from S1/env/3 and by Pepscan analysis. The two methods gave similar results, identifying two epitopes involving amino acids corresponding to residues 740 to 743, and to residues 745 to 750 of gp41. Mutations selected in the chimera with S1/env/3-specific MAbs are identical or similar to changes occurring in vivo in natural isolates of HIV-1. This finding suggests that the epitope may be significant in the neutralization of HIV in vivo.
The reactivity of D antigen-specific monoclonal antibodies with infectious virus and inactivated virus in commercial poliovaccines from two manufacturers was examined in capture enzyme immunoassays. Epitopes on inactivated poliovirus types 2 and 3 in vaccines were modified by the inactivation process to such an extent that some monoclonal antibodies which bound to the homologous infectious virus strain failed to bind to the inactivated virus in the vaccine. This was especially the case for antibodies specific for site 1, which elicits the major immune response to these serotypes in mice. Other epitopes were partially destroyed resulting in a reduction in reactivity of monoclonal antibodies with inactivated virus in vaccines compared to infectious virus. These studies indicate that inactivated virus in vaccine differs antigenically from native virus so that measuring the potency of vaccine in assays in vitro may require careful selection of an appropriate antibody. In addition it is possible that some of the antibodies produced by recipients of formalin-inactivated polioviruses may not contribute to the protective immune response.
Twenty-six laboratories including 22 Blood Transfusion Centres in England, Scotland, Wales and Northern Ireland participated in a collaborative study to assess the suitability of proposed working standards for hepatitis B surface antigens (HBsAg) for use in the UK Blood Transfusion Service. Data from 88 assays indicated that all laboratories could detect HBsAg in the proposed working standard at a concentration of 0.5 IU/ml. Samples containing HBsAg at a concentration of 0.125 IU/ml were scored positive in 80% of assays and it was concluded that this would be a suitable concentration for inclusion in a monitor sample which would indicate whether the sensitivity of an assay system in routine use is varying over a period of time. The use of a batch of 'provisional' working standard tested under field conditions over a period of 4 months has been monitored. HBsAg was not detected in only 2 out of 6,523 assays.
The long-term patency of arteriovenous (AV) fistulas created for hemodialysis of renal-failure patients is usually measured in months, particularly when polytetrafluoroethylene (PTFE) material is interposed between the artery and vein. This is due to the rapid development of intimal hyperplastic lesions in the anastomosis region of the PTFE graft material with the vein. We studied the proliferative patterns in seven human AV fistulas removed at the time of fistula revision. Cell proliferation was determined by using an antibody to the proliferating cell nuclear antigen (PCNA), and specific cell types were identified by immunochemical reagents for smooth muscle cells, monocytes/macrophages, monocytes, lymphocytes, and endothelial cells. All venous segments exhibited a markedly hyperplastic intima. Vascularization of the intima and media by capillary-sized vessels was found. The main intimal cellular component was smooth muscle. Macrophages were usually seen around microvessels, and many also populated the perigraft region of the adventitia. In contrast to human atherosclerotic lesions, high rates of cell proliferation were observed in these fistulas. PCNA indices (percentage of cells that were PCNA positive [mean +/- SD]) were as follows: intima 17.7 +/- 11.3%, media 24 +/- 11.2%, and adventitia 20 +/- 11.6%. However, the distribution of PCNA-positive cells was not uniform. Instead, the PCNA index in microvessel-containing intimal fields was five to six times that of avascular fields (28.9 +/- 10.6% versus 4.9 +/- 4.5%, respectively, p < 0.001). Double immunolabeling revealed a large proportion of PCNA-positive microvascular endothelial cells and surrounding pericyte-like smooth muscle cells, as well as smooth muscle cells without visual connection to either microvessels or the lumen.(ABSTRACT TRUNCATED AT 250 WORDS)
On the basis of animal models of arterial injury, smooth muscle cell proliferation has been posited as a dominant event in restenosis. Unfortunately, little is known about this proliferation in the human restenotic lesion. The purpose of this study was to determine the extent and time course of proliferation in primary and restenotic coronary atherectomy-derived tissue. Primary (n = 118) and restenotic (n = 100) coronary atherectomy specimens were obtained from 211 nonconsecutive patients. Immunocytochemistry for the proliferating cell nuclear antigen (PCNA) was used to gauge proliferation in the atherectomy specimens. The identity of PCNA-positive cells was then determined using immunohistochemical cell-specific markers. Eighty-two percent of primary specimens and 74% of restenotic specimens had no evidence of PCNA labeling. The majority of the remaining specimens had only a modest number of PCNA-positive cells per slide (typically < 50 cells per slide). In the restenotic specimens, PCNA labeling was detected over a wide time interval after the initial procedure (eg, 1 to 390 days), with no obvious proliferative peak. Cell-specific immunohistochemical markers identified primary and restenotic PCNA-positive cells as smooth muscle cells, macrophages, and endothelial cells. In conclusion, the findings were as follows: (1) Proliferation in primary and restenotic coronary atherectomy specimens, as indicated by PCNA labeling, occurs infrequently and at low levels. (2) The response to injury in existing animal models of angioplasty may follow a very different course of events from the clinical reality in human atherosclerotic coronary arteries and may help explain why current approaches to restenosis therapy have been ineffective.
Endothelial attachment is the initial step in leukocyte recruitment into developing atherosclerotic lesions. To determine whether vascular cell adhesion molecule-1 (VCAM-1) expression may play a role in inflammatory cell recruitment into human atherosclerotic lesions, immunohistochemistry was performed with a polyclonal rabbit antisera, raised against recombinant human VCAM-1, on 24 atherosclerotic coronary plaques and 11 control coronary segments with nonatherosclerotic diffuse intimal thickening from 10 patients. Immunophenotyping was performed on adjacent sections to identify smooth muscle cells, macrophages, and endothelial cells. To confirm VCAM-1-expressing cell types, double immunostaining with VCAM-1 antisera and each of the cell-specific markers and in situ hybridization were performed. All atherosclerotic plaques contained some VCAM-1, compared to 45% of control segments. VCAM-1 was found infrequently on endothelial cells at the arterial lumen din both plaques (21%) and in control segments (27%), but was prevalent in areas of neovascularization and inflammatory infiltrate in the base of plaques. Double immunostaining and in situ hybridization confirmed that most VCAM-1 was expressed by subsets of plaque smooth muscle cells and macrophages. The results document the presence of VCAM-1 in human atherosclerosis, demonstrate VCAM-1 expression by human smooth muscle cells in vivo, and suggest that intimal neovasculature may be an important site of inflammatory cell recruitment into advanced coronary lesions.
BACKGROUND: Nerve growth factor (NGF) has been proposed to be critical to normal renal development in rodents. However, little is known about expression of NGF or its receptors in human kidneys, or their potential function in development or disease. EXPERIMENTAL DESIGN: A previously characterized monoclonal antibody (NGFR 5) was utilized for immunohistochemical localization of the p75 NGF receptor (NGFR) in alcohol-fixed tissue sections of human fetal kidney (N = 27, 54 to 105 days gestation), normal mature kidney obtained from nephrectomies for neoplasia (N = 7), and renal biopsies (N = 54) with various glomerulopathies previously characterized for degree of mesangial alpha smooth muscle actin (alpha SM) expression. A second monoclonal antibody (NGFR2) was also utilized on fetal and normal kidney. Immunohistochemical localization of alpha SM and proliferating cell nuclear antigen expression was also performed. RESULTS: Glomerular expression of NGFR in the fetus is limited to the mesangium in later stages of glomerulogenesis; at these stages this expression is similar to that which has been previously reported for platelet-derived growth factor receptor-beta and alpha SM. There is focal, weak persistence of NGFR in normal adult glomeruli, similar to alpha SM. In renal biopsies, glomerular NGFR expression was upregulated in a variety of disease states, which frequently but not invariably correlated with alpha SM expression. Fetal and adult expression of NGFR is also prominent in periarterial connective tissue cells and nerve. Apparent de novo expression by many interstitial cells in normal and diseased adult kidneys is also present. CONCLUSIONS: These studies indicate: (a) NGF or other neurotrophins that bind NGFR may be important in human kidney development and glomerular response to injury; (b) NGFR is a marker of developing mesangial cells similar to alpha SM and platelet-derived growth factor receptor-beta; (c) enhanced expression of NGFR, like alpha SM, is a marker of mesangial cell injury or activation, and that their coordinate upregulation in some glomerular disease states appears to recapitulate a normal developmental state; (d) a population of NGFR and platelet-derived growth factor-beta expressing interstitial cells can be identified in normal kidneys, which suggests potential signaling mechanisms to recruit or activate these cells at sites of tubulointerstitial injury.
The World Health Organization has called for improvements to oral polio vaccines (OPV), particularly the type 3 component. Our improved understanding of the genetic basis of attenuation and antigenicity of this strain gives rise to possibilities for constructing new derivatives via mutagenesis of infectious cDNA. This article reviews progress made with various approaches to constructing a new type 3 candidate vaccine strain. These include the construction of antigen chimaeras of poliovirus based on the Sabin type 1 strain, the introduction of new genetically stable attenuation mutations into the 5' non-coding region (NCR) of the type 3 poliovirus genome, and the introduction of mutations which may increase the thermostability of the type 3 vaccine.
Kinetoplast DNA is a network of interlocked minicircles and maxicircles. In situ hybridization, using probes detected by digital fluorescence microscopy, has clarified the in vivo structure and replication mechanism of the network. The probe recognizes only nicked minicircles. Hybridization reveals prereplication kinetoplasts (with closed minicircles), donut-shaped replicating kinetoplasts (with nicked minicircles on the periphery and closed minicircles in the center), and postreplication kinetoplasts (with nicked minicircles). Replicating kinetoplasts are associated with two peripheral structures containing free minicircle replication intermediates and DNA polymerase. Replication may involve release of closed minicircles from the center of the kinetoplast and their migration to the peripheral structures, replication of the free minicircles therein, and then peripheral reattachment of the progeny minicircles to the kinetoplast.
Fluorescent in situ hybridization with chromosome specific probes was used in conjunction with laser scanning confocal microscopy to assess the three-dimensional distribution of chromosomes in human T-lymphocyte nuclei. Cells in the G1-phase of the cell cycle exhibit a distinctly non-random chromosome organization:centromeric regions of the ten chromosomes examined are localized on the nuclear periphery, often making contact with the nuclear membrane, while telomeric domains are consistently localized within the interior 50% of the nuclear volume. Chromosome homolog pairing is not observed. Transition from the G1 to G2 cell cycle phase is accompanied by extensive chromosome movement, with centromeres assuming a more interior location. Chromosome condensation and chromatin depleted areas are observed in a small subset of G2 nuclei approaching mitosis. These results demonstrate that dynamic chromosome rearrangements occur in non-mitotic nuclei during the cell cycle.
We evaluated the antigenic characteristics of chimeric picornaviruses created by inserting peptide sequences from hepatitis A virus (HAV) capsid proteins into the B-C loop of VP1 of Sabin strain type 1 poliovirus (PV-1). Fifteen viable chimeras were generated. Each retained the ability to be neutralized by polyclonal PV-1 antisera. Two chimeras (H15 and H2) stimulated production of low levels of HAV neutralizing antibodies in immunized rabbits or mice, although in both cases only a small fraction of immunized animals produced this response. The H15 chimera, which contains residues 13-24 of HAV VP1, elicited HAV neutralizing antibodies in three of nine rabbits and at least one of seven immunized mice. These results indicate that a neutralization domain exists in this region of VP1. However, human sera with high titers of antibodies to HAV failed to neutralize or immunoprecipitate this chimera, suggesting the absence of a significant antibody response to this neutralization domain following natural infection. Sera from rabbits immunized with H15 that did not develop HAV neutralizing antibodies contained antibodies reactive with the HAV peptide segment expressed by the H15 virus, indicating substantial differences in the specificities of antibodies elicited by this peptide segment among individual immunized rabbits. The H15 peptide insert was an effective antigen, as indicated by a high level of sensitivity of the H15 chimera to neutralization by a related anti-peptide antibody which was itself devoid of HAV neutralizing activity. One of 16 rabbits immunized with the H2 chimera (residues 101-108 of HAV VP1) developed HAV neutralizing antibodies, confirming both the presence and the highly conformational nature of a neutralization antigenic site involving these residues of HAV.
A collaborative study was carried out to establish a replacement for the International Standard for Rabies Vaccine, the stocks of which are exhausted. Three rabies vaccines for human use derived from different rabies virus strains and prepared on different cell culture substrates were compared with the International Standard for Rabies Vaccine using in vivo and in vitro assay methods in a collaborative study involving 14 participants. The proposed fifth International Standard (PISRAV) which was derived from the same virus strain as the present international standard preparation, the Pitman Moore (PM) strain, was found to be approximately twice as potent relative to the International Standard in immunogenicity assays as in antigenicity assays. On the other hand another vaccine, derived from the LEP strain, was considerably more potent in antigenicity assays than in immunogenicity assays. The glycoprotein of the proposed replacement standard measured in antigenicity assays appeared to be stable at +37 degrees C for 245 days, whereas the immunogenicity of the proposed replacement vaccine was sensitive to this heat treatment and the vaccine lost 66% of its immunogenic potency. The results of this study indicate that the NIH protection test should continue to form the primary basis for potency assay of rabies vaccine as glycoprotein content does not appear to correlate with immunogenic potency for different types of vaccine. The vaccine coded PISRAV has been established as the fifth International Standard for Rabies Vaccine and a potency of 16 International Units of Rabies Vaccine (based on the immunogenicity assays) assigned to the contents of each ampoule. Each ampoule has also been assigned a unitage of 10 IU of PM Rabies Virus Glycoprotein and 135 IU of PM Rabies Virus Ribonucleoprotein.
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We have raised antisera and monoclonal antibodies (MAbs) to the C4 region of HIV-1 gp120, using an antigen chimaera of poliovirus as immunogen. These MAbs and sera, together with MAbs to the same region raised by other methods, fall into three groups defined by their abilities to bind to recombinant gp120 and/or the immunogenic peptide. In some cases, the amino acids recognized by the MAbs have been identified by pep-scan and by solution phase peptide inhibition of binding to recombinant gp120. Our results indicate that the amino acids WQEVGKAMYA are exposed on the surface of recombinant gp120. Antibodies to these amino acids on recombinant gp120 compete for soluble CD4 binding in vitro, but only weakly neutralize HIV.
Lipoprotein lipase (LPL), hydrolyzes the core triglycerides of lipoproteins, thereby playing a role in their maturation. LPL may be important in the metabolic pathways that lead to atherosclerosis, since it is secreted in vitro by both of the predominant cell types of the atherosclerotic plaque, i.e., macrophages and smooth muscle cells. Because of uncertainty concerning the primary cellular source of LPL in atherosclerotic lesions, in situ hybridization assays for LPL mRNA were performed on 12 coronary arteries obtained from six cardiac allograft recipients. Macrophages and smooth muscle cells were identified on adjacent sections with cell-specific antibodies and foam cells were identified morphologically. LPL protein was localized using a polyclonal antibody. LPL mRNA was produced by a proportion of plaque macrophages, particularly macrophage-derived foam cells, but was not detected in association with any intimal or medial smooth muscle cells. These findings were confirmed by combined immunocytochemistry and in situ hybridization on the same tissue sections. LPL protein was detected in association with macrophage-derived foam cells, endothelial cells, adventitial adipocytes, and medial smooth muscle cells, and, to a lesser extent, in intimal smooth muscle cells and media underlying well-developed plaque. These results indicate that macrophage-derived foam cells are the primary source of LPL in atherosclerotic plaques and are consistent with a role for LPL in the pathogenesis of atherosclerosis.
Tissues fixed with organic solvent fixatives such as Carnoy's solution are known to give poor and erratic results with in situ hybridization, whereas those fixed with paraformaldehyde produce more consistent results. To understand this difference and to improve the utility of Carnoy's-fixed tissue for in situ hybridization, we explored several parameters of RNA integrity and preservation. Carnoy's-fixed, paraffin-embedded livers and paraformaldehyde-fixed, paraffin-embedded livers of mice were compared for RNA extractability, degradation, and hybridizability. In addition, retention of RNA in tissue sections after sequential in situ hybridization treatments was compared. RNA was found to be easily extractable from Carnoy's-fixed liver and was well preserved, with only slight degradation of high molecular weight RNA. Conversely, only a small percentage of the RNA was extractable from paraformaldehyde-fixed liver unless the tissue was digested with protease. The extracted RNA was well preserved, without detectable degradation. Sections of tissue fixed in Carnoy's solution subjected to in situ hybridization retained only about 10% of their original RNA content and gave correspondingly weak in situ hybridization signals. Formaldehyde-fixed tissues retained much more of the RNA (about 45%) and produced strong in situ hybridization signals. Treatment of Carnoy's-fixed tissue sections with vaporous formaldehyde increased retention of RNA and provided in situ hybridization signals comparable with those of paraformaldehyde-fixed tissues.
Funeral cost was less for younger people and for cremation. Among 156 funerals examined, after matching for sex, age, and cremation/burial, the funerals of a small sample of 6 suicides cost less than those of the controls.