Autoimmune disease: etiology, therapy and regeneration.
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Autoimmune Diseases↗
Biomedical subjects
M Peakman
Publications and source records attributed to M Peakman.
Relationship between the tumour-associated antigen 90K and cytokines in the circulation of persons infected with human immunodeficiency virus.
A tumour-associated antigen known as 90K has been found in high concentrations in the serum of patients infected with human immunodeficiency virus (HIV) even in the absence of neoplastic complications. In order to investigate the relationship between the production of 90K and soluble inflammatory mediators, we studied serum concentrations of the antigen, tumour necrosis factor-alpha (TNF-alpha), interleukin-I-alpha (IL-I-alpha), interferon-gamma (IFN-gamma), IFN-alpha, neopterin and beta 2-microglobulin (beta 2-m) in patients with non-neoplastic HIV infection at various stages of disease and in control persons. The antigen was detected in all those studied but its concentration was higher in HIV-infected patients compared with controls (P < 0.001), increasing progressively with advancing stages of disease. There was a negative correlation between concentrations of 90K and IL-I-alpha in patients in U.S.A. Centers for Disease Control groups II and III (P < 0.02) and also between that of 90K and both TNF-alpha (P < 0.01) and IL-I-alpha (P < 0.05) in control persons. The results indicate that 90K is not merely a tumour-associated antigen and that its production may be part of immune and inflammatory responses in the absence of neoplasia. The correlation between the concentrations of 90K and of some cytokines in asymptomatic patients and healthy persons suggests that 90K may be part of a network of immune and inflammatory reactants.
Adult↗
Development of techniques for obtaining monodispersed human islet cells.
The aim of this study was to develop techniques to obtain monodispersed, human islet cells in large quantities, since these constitute a potentially transplantable beta cell mass with which to treat established type 1 diabetes, as well as provide the most appropriate substrate for studying the immune pathogenesis of the disease. Human islets were isolated from the pancreas of beating-heart organ donors by collagenase digestion. Enzymatic (collagenase types II, IV, V, and XI, trypsin, DNAse, and hyaluronidase) and chemical (EDTA and EGTA) conditions were then used to find the optimum requirements for digestion of intact human islets into their constituent cells. The combination of trypsin with EDTA provided the highest yield of monodispersed islet cells (963 cells/islet) and highest viability (88%). DNAse with EGTA gave high yields (710 cells/islet) but viability was low (55%). Lower yields and viability were obtained using collagenase types II, IV, V, and XI (47-243 cells/islet; viability 45-62%), hyaluronidase (410 cells/islet; 75% viability), and EDTA alone (253 cells/islet; viability 43%). Human islet cells monodispersed using trypsin 0.125 mg/ml in 0.1 mM EDTA retained an insulin secretory response to glucose, and had intact surface class I MHC molecules when analyzed immediately after digestion by flow cytofluorimetry. Our results indicate that functionally intact, single, human islet cells may be obtained in abundance, and provide a potential substrate for islet cell transplantation in the treatment of patients with type 1 diabetes.
Cell Separation↗
Differences in immune recognition of cytochrome P4502D6 by liver kidney microsomal (LKM) antibody in autoimmune hepatitis and chronic hepatitis C virus infection.
LKM-1 antibody, which characterizes a subtype of autoimmune hepatitis (AIH), is also found in some patients with chronic hepatitis C virus (HCV) infection. It has been suggested that HCV initiates autoimmunity through molecular mimicry, because there is partial identity between HCV and cytochrome P4502D6 (CYP2D6), the putative target of LKM-1. Whether CYP2D6 is the target of LKM-1 in HCV-related liver disease, however, is controversial. To clarify this issue, we have studied by phage plaque assay and Western blot the reactivity to recombinant CYP2D6, isolated from a human liver cDNA library, in 55 patients with LKM-1, 18 (14 females, median age 12 years) anti-HCV-negative, with classical AIH, and 37 (27 females, median age 52 years) anti-HCV-positive. Reactivity to CYP2D6 was found in 72% of the anti-HCV-negative, but only in 27% of the anti-HCV-positive patients (P < 0.001), although immunofluorescence LKM-1 titres were similar in the two groups. In addition, to investigate whether the antibody responsible for the LKM-1 fluorescent pattern also reacts with CYP2D6, we have determined the specificity of LKM-1 antibodies present in the supernatant of lymphoblastoid B cell lines obtained from two patients with LKM-1-positive AIH. An oligo/monoclonal antibody thus generated gave both the typical fluorescent pattern and reacted with CYP2D6. Our results show that whilst antibodies producing the characteristic LKM-1 fluorescent pattern can react with CYP2D6, not all LKM-1-positive sera do so, particularly if obtained from patients with chronic HCV infection. This suggests that LKM-1 in HCV infection recognizes epitopes or antigens different from those targeted in AIH.
Adolescent↗
An investigation of antibodies to nerve growth factor in diabetic autonomic neuropathy.
Nerve growth factor (NGF) is required for development and maintenance of autonomic nervous function and is highly expressed in the iris. An association between sensory and autonomic diabetic neuropathy and NGF function has been postulated. In light of the evidence for an autoimmune component to the pathogenesis of autonomic neuropathy in Type 1 diabetes mellitus, an enzyme linked immunosorbent assay (ELISA) was established to investigate the presence of autoantibodies to mNGF in: 20 patients with long-standing Type 1 diabetes with abnormal autonomic function tests, of whom 14 had symptomatic autonomic neuropathy, 3 had an episode of iritis, and 6 had no autonomic symptoms; 9 age-matched patients with Type 1 diabetes and no complications; 10 healthy control subjects. Insulin antibodies by ELISA and autoantibodies to other endocrine targets were also measured. The specificity of anti-mNGF autoantibody ELISA was further confirmed by immunodepletion on mNGF-Sepharose 4B. No differences in antibody binding to mNGF were detected, between any of the patient groups and control subjects. There was no relationship between age, sex, and diabetes duration and mNGF binding. High levels of anti-mNGF autoantibodies were found in only one diabetic patient who had no evidence of neuropathy, raising the possibility that an autoimmune component to NGF might precede the development of autonomic dysfunction. It remains to be established whether autoantibodies to NGF play a role in diabetic autonomic neuropathy and prospective studies will be required to investigate whether the autoantibodies are a feature of evolving but not established neuropathy.
Adult↗
Effect of fresh frozen plasma and gammaglobulin on humoral immunity in neonatal sepsis.
Fresh frozen plasma and intravenous immunoglobulin are used as prophylaxis against, and for the treatment of, neonatal infection. It is assumed that any beneficial effect is mediated through the humoral immune factors contained in each preparation. The effect of fresh frozen plasma and intravenous immunoglobulin on humoral immune markers (immunoglobulins and IgG subclasses, complement components and activation products, and C reactive protein) was investigated over a 24 hour period after their randomised administration to 67 infants with suspected infection. Thirty infants without suspicion of infection were studied as controls. Compared with control infants, infants with suspected infection had increased concentrations of C reactive protein, reduced concentrations of fibronectin, and increased concentrations of the complement activation marker C3d, but similar concentrations of IgG, IgG subclasses, IgA, and IgM. After intravenous immunoglobulin treatment (500 mg/kg) concentrations of total IgG and all IgG subclasses increased, as did IgA and complement component C4. Concentrations of C reactive protein decreased after intravenous immunoglobulin treatment and were significantly lower than baseline after 24 hours. In contrast, no change in IgG or IgG subclass concentrations occurred after fresh frozen plasma administration. At 24 hours after fresh frozen plasma administration, concentrations of IgA, IgM, and C4 were significantly higher than baseline and serum IgA was significantly higher than in infants tested 24 hours after intravenous immunoglobulin treatment. These results confirm the rational basis for intravenous immunoglobulin treatment but question the value of fresh frozen plasma, particularly in the light of its attendant problems as an untreated blood product.
Antibody Formation↗
Increased expression of T-cell markers of immunological memory associated with protection from type I diabetes. A study of identical twins.
Disturbances in the balance of CD4+ helper T-lymphocytes expressing the surface molecules CD45RA and CD45R0, which define naive and memory populations, respectively, are present at diagnosis of type I diabetes. In a prospective study over 10 years, these subsets were analyzed in samples obtained from 18 identical twins of patients with type I diabetes, 8 of whom became diabetic (prediabetic twins), whereas the rest remained nondiabetic after at least 8 years follow-up and are now unlikely to develop the disease (diabetes-protected twins). At the beginning of the study, percentage levels of naive (CD45RA+) CD4+ lymphocytes were significantly elevated in prediabetic twins compared with diabetes-protected twins (P < 0.05) and remained so throughout the study (P < 0.01). Percentage levels of naive cells in diabetes-protected twins were significantly reduced compared with control subjects both at the beginning and throughout the study (P < 0.05, P < 0.01, respectively). In contrast, diabetes-protected twins at the beginning of the study had elevated percentage levels of memory (CD45R0+) CD4+ lymphocytes that persisted throughout the study compared with prediabetic twins (P < 0.05 for both). Percentage levels of memory cells in prediabetic twins were significantly reduced compared with control subjects both at the beginning and throughout the study (P < 0.01, P < 0.05, respectively). Increased percentage levels of a population of CD4+ lymphocytes coexpressing CD45RA and CD45R0 were seen in both twin groups compared with control subjects at entry into and during the study (P < 0.05 for all), but persisted only in the prediabetic twins.(ABSTRACT TRUNCATED AT 250 WORDS)
Adolescent↗
High prevalence of autoantibodies to glutamic acid decarboxylase in long-standing IDDM is not a marker of symptomatic autonomic neuropathy.
Immune reactivity to the enzyme glutamic acid decarboxylase (GAD), a pancreatic islet autoantigen, is present at the diagnosis of insulin-dependent diabetes mellitus (IDDM). Because GAD is also highly expressed in the nervous system, we investigated the presence of autoantibodies to the isoform GAD65 in patients with diabetic neuropathy, which is a debilitating complication of the disease. We studied 39 patients with autonomic and somatic neuropathy, 28 patients matched for age and IDDM duration, and 13 patients with a shorter duration of IDDM, all with no diabetic complications, as well as 50 recently diagnosed diabetic patients, 23 neurologic patients with idiopathic autonomic failure unrelated to IDDM, and 72 healthy subjects. An immunoprecipitation radioligand assay was used to detect anti-GAD65 autoantibodies with in vitro transcribed and translated human islet GAD65 as antigen. Autoantibodies to GAD65 were present in 56% of the diabetic patients with neuropathy, 57% of the long-duration and 69% of the short-duration diabetic control subjects, 78% of the recently diagnosed patients, and 13% of the nondiabetic neuropathic patients. Among the diabetic patients with neuropathy, there was no correlation between the presence of anti-GAD65 antibodies and the presence of autoantibodies to sympathetic ganglia, vagus nerve, or adrenal medulla structures identified by immunofluorescence. Our study shows that anti-GAD65 antibodies are present in a high proportion of patients with diabetic neuropathy but are not exclusively associated with it, rendering it unlikely that they have a role as a disease marker or that they are pathogenetic.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
CD5+ B-cells at the onset of type I diabetes and in the prediabetic period.
OBJECTIVE: B-cells expressing CD5 are associated with the production of autoantibodies and are present at increased levels in several autoimmune diseases. The aim of this study was to investigate the relationship of these cells to the development of type I diabetes and the presence of organ and non-organ-specific autoantibodies. RESEARCH DESIGN AND METHODS: We measured percentage levels of CD5+ B-cells in patients with recent-onset (n = 34) and long-standing (n = 21) type I diabetes and in a cohort of 18 identical twins of patients with type I diabetes studied prospectively, 8 of whom became diabetic (prediabetic twins) during the study; the rest remained nondiabetic after at least 7 years and are now unlikely to develop the disease. Forty-seven healthy individuals were studied as control subjects. RESULTS: Percentage levels of total B-cells (CD20+) and the proportion expressing CD5 were increased in patients with recent-onset (P < 0.001 for both) but not long-standing type I diabetes compared with control subjects. Percentage levels of CD20+ B-cells were increased in prediabetic twins throughout the prediabetic period (P < 0.05), and there was an increased proportion of CD5-expressing B-cells that failed to reach statistical significance (P = 0.08). Percentage levels of CD20+ B-cells and the proportion expressing CD5 were normal throughout the study in twins remaining nondiabetic. No relationship between percentage levels of CD5+ B-cells and islet cell antibody, thyroid autoantibodies, or non-organ-specific autoantibodies was found. CONCLUSIONS: These results show an increase in B-cell percentage levels at the diagnosis of type I diabetes, which is because of an expansion of the CD5+ subset. These changes are also evident in twins throughout the prediabetic period, which suggests that they are related to the processes that lead to diabetes.
Adult↗
T cell clones generated from patients with type 1 diabetes using interleukin-2 proliferate to human islet antigens.
T lymphocytes are implicated in the pathogenesis of Type 1 (insulin dependent) diabetes. Activated T lymphocytes expressing IL-2 receptors are found at increased levels in the peripheral blood in the prediabetic period, at diagnosis and for several months after the onset of the disease, but their role in the pathogenesis of the disease is not known. We have used co-culture of peripheral blood lymphocytes with IL-2 alone to selectively generate T cell clones from the in vivo activated T cell population, and examined the phenotype and antigen specificity of the clones derived. From 3 patients with newly-diagnosed Type 1 diabetes 184 clones were generated, the majority of which (39%) were CD4+TCR alpha beta+, whilst 31% were CD8+TCR alpha beta+. From 2 healthy control subjects 90 clones were obtained, of which 62% were CD4+TCR alpha beta+ and 33% were CD8+TCR alpha beta+. Antigen specificity was examined in 46 clones from the patients and 44 from the control subjects in proliferation assays, using as antigens homogenate of human islets of Langerhans, human islet membrane preparation and human liver membrane preparation. Three clones (all CD4+TCR alpha beta+) from the patients, but none from the control subjects, proliferated in a dose dependent fashion in response to stimulation with human islet homogenate presented by autologous APCs, but to neither of the other autoantigen preparations. Our results demonstrate that a relatively high proportion (7%) of T lymphocytes activated in vivo recognise human islet antigens, indicating that they may have a role in the pathogenesis of the disease.
Adult↗
Analysis of peripheral blood mononuclear cells in rodents by three-colour flow cytometry using a small-volume lysed whole blood technique.
Techniques were adapted to permit phenotypic analysis of peripheral blood leucocytes in rats and mice using small volumes of lysed whole blood and single, dual- or triple-colour immunofluorescence staining with flow cytometry. Red cell lysis using proprietary lysing solutions provides populations of lymphocytes, monocytes and granulocytes clearly differentiable by their light scatter properties. In the present study, measurement of functional and activation T lymphocyte subsets was performed by single- and dual-colour flow cytometry in both species and in excess of 5000 lymphocytes could be counted using as little as 10 microliters of whole blood. Triple-colour fluorescence, using antibodies labelled with fluorescein, phycoerythrin and biotin permitted simultaneous detection of three phenotypic markers. This novel technique has two major advantages, maximising the phenotypic information which may be obtained and using volumes of blood which are sufficiently small that they can be drawn regularly from the same animal.
Animals↗
Methods for assessing complement activation in the clinical immunology laboratory.
Complement activation is a key component of the pathogenesis of immune-mediated tissue damage in many diseases. Assessment of complement activation in current practice is largely based on the measurement of intact C3 and C4 or the determination of complement haemolytic function. These parameters reflect activation only indirectly, are insensitive and open to influence by factors other than complement conversion. New approaches to evaluate complement activation directly using sensitive techniques have been developed, and several could be adopted easily in most laboratories. These concentrate on the detection of activation fragments, neoantigens or complexes that only arise as a direct result of complement activation. The wide application of these techniques in research and clinical practice would enhance our understanding of the pathogenesis of a range of inflammatory and infectious diseases.
Anaphylatoxins↗
Case against subclassification of type II autoimmune chronic active hepatitis.
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Adolescent↗
Autoantibodies to nervous tissue structures are associated with autonomic neuropathy in type 1 (insulin-dependent) diabetes mellitus.
There is evidence that the immune system may play a role in the pathogenesis of autonomic neuropathy in Type 1 (insulin-dependent) diabetes mellitus. In the present study, we investigated the presence of autoantibodies to sympathetic and parasympathetic nervous structures and their correlation with other conventional autoantibodies in well-characterised diabetic populations, with or without diabetic neuropathy, and normal subjects. An indirect immunofluorescent complement-fixation technique was used, with monkey adrenal gland, rabbit cervical ganglia and vagus nerve as substrates. Of the patients with symptomatic autonomic neuropathy 33% were positive for at least one autoantibody (20% anti-sympathetic ganglia, 10% anti-vagus nerve and 13% anti-adrenal medulla). The frequency of having one or more antibodies to nervous tissues and the prevalence of anti-cervical ganglia antibodies were significantly higher in the neuropathic patients than in the diabetic control subjects with disease of similar duration and in the normal subjects (p < 0.05). Of the patients without complications with diabetes of shorter duration 33% were also positive for at least one autoantibody (13% anti-ganglia, 13% anti-vagus nerve and 13% anti-adrenal medulla). No correlation was found with other tissue autoantibodies, including islet cell antibodies. Our data indicate that nervous tissue autoantibodies are associated with symptomatic autonomic neuropathy. Anti-sympathetic ganglia and anti-vagus nerve antibodies seem to be more disease-specific. Patients with diabetes of shorter duration who were positive for these autoantibodies may represent pre-neuropathic patients.
Adolescent↗
T cell activation and disease severity in HIV infection.
In vitro studies have indicated that T lymphocyte activation may be of importance in the pathogenesis of HIV infection. In order to define the role of immune activation in vivo, we assessed the expression of the T cell activation markers HLA-DR and CD25 by flow cytometry in peripheral blood in relation to disease severity and the surrogate markers CD4 and beta 2-microglobulin in 157 patients with HIV infection and 53 healthy seronegative blood donors. Percentage levels of CD3+HLA-DR+ T lymphocytes were significantly higher (P < 0.0001) and percentage levels of CD3+CD25+ T lymphocytes significantly lower (P < 0.0001) in all HIV+ patients compared with controls. A significant correlation was observed between increasing percentage levels of CD3+HLA-DR+ T lymphocytes and both declining CD4 counts (r = 0.52; P < 0.001) and increasing beta 2-microglobulin levels (r = 0.56; P < 0.001). Percentage levels of CD4+HLA-DR+ and CD4+ CD25+ lymphocytes were significantly higher in all HIV+ patients compared with controls (P < 0.001). Levels of activated (HLA-DR+ and CD25+) CD4+ lymphocytes showed a significant step-wise linear increase with increasing disease severity (P < 0.001). High levels of CD3+HLA-DR+ T lymphocytes were found in a greater proportion (81.8%) of asymptomatic HIV+ patients (Centres for Disease Control (CDC) group II) than low CD4 counts (51.5%) (P < 0.001). Compared with controls, HIV+ patients had higher percentage levels of CD8+HLA-DR+ lymphocytes (P < 0.001), but similar levels of CD8+CD25+ lymphocytes. These results indicate that T cell activation is not only a consistent but also an early feature in HIV infection. Monitoring levels of activated T cells and their subsets is of value in assessing progression of HIV-related disease.
Adult↗
Immunological studies on type 1 diabetes in identical twins.
Diabetes is a multifactorial disease, the pathogenesis of which involves participation of the host immune system in beta cell destruction. Studies on identical twins offer the opportunity to define genetic and non-genetic factors which may contribute to susceptibility to the disease. Our own work indicates that the number and nature, as well as the intensity and persistence of immune abnormalities of cellular and humoral immune responses can be powerful predictors of the disease and also identify twins who have a degree of protection from the disease.
Antibody Formation↗
Increase in simultaneous coexpression of naive and memory lymphocyte markers at diagnosis of IDDM.
The monoclonal antibodies 2H4 (anti-CD45RA) and UCHL1 (anti-CD45RO) were used to subdivide the CD4 and CD8 T-cell subsets into naive and memory cells. The peripheral blood lymphocytes of 34 patients with recent-onset IDDM, 21 patients with long-standing IDDM, and healthy control subjects of similar age and sex were analyzed by a three-color immunofluorescence technique. CD4 and CD8 lymphocytes expressed the CD45 isoforms alone (CD45RA+ or CD45RO+) or in combination CD45RA+RO+). Simultaneous coexpression of both CD45RA and CD45RO (CD45RA+RO+) on CD4 and CD8 lymphocytes in patients with recent-onset IDDM was higher than in control subjects (P < 0.001). The proportion of CD4 lymphocytes expressing CD45RA alone (CD45RA+RO-) was similar in these groups, but the percentage of CD8 lymphocytes that were CD45RA+RO- was significantly higher in the patients with recent-onset IDDM (P < 0.05). The result of these changes is a significant increase in expression of naive phenotypes (CD45RA+ and CD45RA+RO+) on CD4 and CD8 lymphocytes in recent-onset IDDM (P < 0.005 and P < 0.0001). In long-standing IDDM, total CD45RA+ expression on CD4 and CD8 lymphocytes was reduced compared with control subjects (P < 0.05) as a result of a tendency of CD45RA+RO- and CD45RA+RO+ subsets to be lower. This increase in total naive (CD45RA+) lymphocytes and in coexpression of naive (CD45RA) and memory (CD45RO) markers on CD4 and CD8 lymphocytes subsets in patients with recent-onset IDDM suggests that abnormal regulation of T-cell activation and maturation is important in the pathogenesis of the disease.
Adult↗
Lymphocyte vaccination prevents spontaneous diabetes in the non-obese diabetic mouse.
The aim of this study was to investigate whether lymphocyte vaccination can prevent diabetes occurring in the non-obese diabetic (NOD) mouse, an animal model of human insulin-dependent diabetes mellitus (IDDM). The lymphocyte vaccine was composed of lymphocytes isolated from the spleens of diabetic NOD mice, activated in vitro using concanavalin A (Con A) and rendered immunogenic using glutaraldehyde treatment. These cells were used to vaccinate mice at 6 weeks with boosters at weeks 10, 14 and 18. The animals were then monitored for signs of diabetes until week 30. Twenty-eight NOD mice (11 male, 17 female) were T-lymphocyte vaccinated while 35 littermates (14 male, 21 female) were sham vaccinated with the vaccine carrier, as control mice. The percentage of mice remaining non-diabetic was 50% in the T-lymphocyte-vaccinated group compared with 20% in control mice (P < 0.05). When the results were divided according to sex of the mouse the percentage of female NOD mice remaining non-diabetic was 47.1% in the T-lymphocyte-vaccinated group compared to only 9.4% in the controls (P < 0.01), while in the males there was no significant difference between the groups. These results suggest that T-lymphocyte vaccination can prevent diabetes in NOD mice and that it has its greatest effect in females. The therapy is apparently safe and its efficacy indicates that it may be of value in prediabetes in man.
Animals↗