Hypothyroidism and anti-endothelial cell antibodies.

Because of our previous demonstration of anti-endothelial cell antibodies (AECA) in patients with insulin-dependent diabetes mellitus and their association, in this condition, with thyroid disease, we sought these antibodies in patients with suspected thyroid dysfunction using an enzyme immunoassay with human umbilical vein endothelial cells as the substrate. AECA were found in 5/120 (4.2%) patients with normal and 15/97 (15.4%) with abnormal thyroid function. The increased prevalence in the latter group was due to a highly significant association between the presence of AECA and raised levels of TSH. We conclude that a highly significant correlation exists between the levels of AECA and TSH, but not between those of AECA and fT4. Patients with hypothyroidism as defined by high levels of TSH have AECA significantly more often than patients with low or normal TSH (22.2% versus 2.8% and 5.8%).

Anti-endothelial cell antibodies in nephropathia epidemica and other viral diseases.

Increased capillary permeability is a central feature of the severe forms of haemorrhagic fever with renal syndrome (HFRS) and occurs also, though less frequently, in nephropathia epidemica (NE), one of the milder forms of this syndrome, caused by Puumala virus. We therefore searched for antiendothelial cell antibodies (AECA) in patients with NE and in those with other presumed or serologically proven acute viral illnesses. By enzyme immunoassay, using human umbilical vein endothelial cells (HUVEC) as the substrate, IgG class AECA were detected significantly more frequently in patients with NE and with influenza A than in Red Cross blood donors. A lesser degree of reactivity could be shown with a human alveolar cell carcinoma line and with human and mouse embryonic fibroblasts. Pretreatment of HUVEC with interferon-gamma (IFN-gamma), but not with IL-1 or tumour necrosis factor-alpha (TNF-alpha), increased their ability to bind IgG of sera from patients with NE and acute febrile illnesses. We conclude that, although AECA can be demonstrated in NE, they occur also in other acute viral illnesses and, unless cytopathic by a mechanism not shared by the AECA of these other illnesses, are unlikely to be casually related to the capillary leak in HFRS.

Anti-endothelial cell antibodies in insulin-dependent diabetes mellitus.

The prevalence of IgG class antibodies to endothelial cells (AECA) was studied in 136 young patients with insulin-dependent diabetes mellitus by an enzyme immunoassay using human umbilical cord vein endothelial cells. The patients were divided into four groups according to the time between diagnosis and study and their results were compared with those in control children and blood donors. AECA became progressively more frequent with the duration of diabetes, being 4% in diabetics tested within 2 weeks of diagnosis and reaching 34% after an average disease duration of 11.2 years. They were not more common in patients with neuropathy, retinopathy or nephropathy than in patients without these complications, but were associated with co-existing thyroid disease and IgA deficiency. The results suggest that in insulin-dependent diabetes mellitus AECA are associated with co-existing autoimmune disorders but not with diabetic microvascular disease.

Endothelial cell activation in vasculitis of peripheral nerve and skeletal muscle.

To clarify the role of endothelial cells in the pathogenesis of vasculitis affecting peripheral nerve and skeletal muscle, the endothelial expression of adhesion molecules and major histocompatibility antigens (MHC) in different vasculitic syndromes were studied, and related to the presence of anti-endothelial cell antibodies (AECA). Increased expression of the intercellular adhesion molecule ICAM-1 in vasculitic lesions in nerve and muscle was shown, and this was associated with increased expression of MHC class I and II antigens. AECA were detected in low titre in only a minority of patients. The findings suggest that endothelial cells have a critical role in mediating the tissue injury in vasculitis affecting nerve and muscle and that the process is triggered by cellular and not antibody-mediated mechanism in the majority of patients.

Polyclonal B-cell activation after intravenous immunoglobulin therapy in idiopathic thrombocytopenic purpura--an anti-idiotype response?

Polyclonal B cell activation occurred in 3 patients following treatment with intravenous immunoglobulin (i.v. Ig) for idiopathic thrombocytopenic purpura (ITP). The possibility that this may represent an anti-idiotype response and the hypothesis that prolonged remission of ITP may be induced by this mechanism are discussed.

Effects of a human urinary mitogen on subpopulations of peripheral blood mononuclear leukocytes.

We have earlier isolated, to apparent homogeneity, a 27-28 kD human basic protein (UM) from the urine of a patient with myelomonocytic leukaemia. UM is a mitogen for resting human peripheral blood mononuclear leukocytes (PBML). We have now further defined the effect of UM on human PBML and their subpopulations in 6-day cultures. Cell proliferation was measured by 3H-thymidine uptake and Ig production by the plaque forming cell (PFC) response. Whole PBML responded to UM with proliferation and an increase in PFC. The PFC response was at best equal to and frequently synergistic with that produced by pokeweed mitogen and occurred in the three major Ig classes. To test the effect of UM on subpopulations of PBML, adherent cells (AC) were isolated by plastic adherence and T and B enriched populations by rosetting with sheep red blood cells. The proliferative response of T cells needed the presence of AC whilst the effect on Ig production by B cells required both T cell help and the presence of AC. Human thymocytes also responded to UM by proliferation. The results show that, in addition to being a T cell mitogen, UM is also a T cell dependent polyclonal B cell activator.

Suppressor factors generated from human mononuclear cells by means of purified myeloma proteins.

Peripheral blood mononuclear cells (PBMC) from normal human donors were cultured in Marbrook flasks in the presence of purified IgG or IgA myeloma proteins. The culture supernatants were tested for their ability to suppress pokeweed mitogen (PWM)- or Epstein-Barr virus (EBV)-driven Ig synthesis by normal PBMC. Two supernatants from PBMC cultured with IgG and one from PBMC cultured with IgA were tested and suppressed PWM-driven Ig synthesis as measured by a reverse haemolytic plaque assay and by quantitation of the Ig secreted into the culture medium of the PWM-driven cells. This suppression was not restricted to the Ig isotype of the 'inducing' myeloma protein, but was extended to IgG, IgA, and IgM. The suppressive effect could be absorbed out with human IgG.

Hypophosphatemic rickets, hyperparathyroidism, and hyperthyroidism with hypercalcemia.

We describe a case of hypophosphatemic rickets in which hyperparathyroidism and hypercalcemia developed and were possibly secondary to co-existent hyperthyroidism. During the period of hypercalcemia with high immunoreactive parathyroid hormone levels the serum inorganic phosphorus level became, and remained, normal. This supports the concept that renal tubular phosphate absorption may be sensitive to calcium.

The effect of phenytoin in vitro on normal human mononuclear cells and on human lymphoblastoid B cell lines of different Ig isotype specificities.

The anticonvulsant drug phenytoin, in less than cytotoxic concentrations, caused significant reductions in Ig secretion by unstimulated or EBV-stimulated normal MNC, as measured by PFC or secretion of Ig into the culture medium. Isotype-specific LBL varied in their sensitivity, the secretion of IgA (1 line) and IgG (3 lines) being reduced by phenytoin near therapeutic concentrations, whereas that of IgM (1 line) was resistant. Six-day exposure of MNC to phenytoin caused no selective depletion of or enrichment for B cells, monocytes or T cell subsets. The results suggest that the reduction in serum Ig levels reported in phenytoin-treated epileptic patients is, at least in part, due to a direct effect of the drug on the B lymphocyte. However, among EBV-activated normal MNC, those secreting IgA were no more sensitive to the drug than those secreting IgG or IgM, and other factors may, therefore, operate to cause the preferential reduction in serum IgA in phenytoin-treated patients.

Polyclonal B-cell activation and increased lymphocyte helper-suppressor ratios in discoid lupus erythematosus.

We studied polyclonal B-cell activation in twenty-six patients with discoid lupus erythematosus (DLE). Spontaneous plaque-forming cells of the IgA class (IgA-SPFC) as determined by a reverse haemolytic plaque assay were significantly more common in patients with DLE than in fifty control subjects. The patients showed a positive correlation between IgA-SPFC and OKT4/8 ratios and also had a significantly higher mean OKT4/8 ratio. The two groups did not differ with regard to cells producing IgG or IgM or cells with OKT3, OKT4, OKT8 or OKMI markers. None of the three patients with DLE who had IgA-SPFC values which were above the mean (+2 s.d.) for the control subjects had positive tests for ANA or low serum C3 or C4, but two of the three also had increased IgG-SPFC values. The results indicate that polyclonal B-cell activation occurs in a small proportion of patients with DLE.

A comparison of three reverse haemolytic plaque assays.

One fluid phase and two solid phase reverse haemolytic plaque (RHP) assays were used to quantitate plaque forming cells (PFC) in pokeweed mitogen (PWM) stimulated and unstimulated cultures of mononuclear cells (MNC) from 18 normal donors. There was a close correlation between the results of the three assays and between each one and the number of cells containing cytoplasmic Ig (cIg). All three correctly identified donors with a low PWM response. The fluid phase assay was the most sensitive with PFC values in PWM stimulated cultures two to four times higher than with the solid phase assays.

Antibody profiles of sera giving different nuclear staining patterns.

Antibodies to three major antigens of the non-histone or saline-extractable nuclear antigen (ENA) complex were sought by counterimmunoelectrophoresis (CEP) in three groups of sera which gave different patterns in the immunofluorescence test for antinuclear antibodies (ANA). Precipitins, mainly anti RNP and anti SS-B, were found most commonly (61%) in 70 sera with a speckled ANA pattern but were less frequent (8%) in 61 sera with a homogeneous ANA pattern and exceptional (1%) in 72 sera which showed fibrillar ANA staining. Rim staining was an insensitive indicator of nDNA antibody. An enzyme immunoassay (EIA), specific for anti-SS-B was more sensitive than CEP and identified this antibody in 28 sera, compared with 18 for CEP.

Characteristics of human mononuclear cell fractions separated on the basis of theophylline sensitivity of E rosette forming ability.

T lymphocytes may be separated into theophylline-sensitive (Tsens) and theophylline-resistant (Tres) fractions based on their loss or retention of ERFC-forming ability after incubation with 3 mM theophylline. It has been claimed that Tsens cells have a predominant suppressor function, whilst Tres cells have mainly helper activity. We have studied the sensitive and resistant fractions to ascertain their degree of homogeneity with regard to putative markers for helper and suppressor function. Incubation with theophylline caused no significant change in the expression of OKT3, OKT4, OKT8, and OKM1 antigens. Compared to Tsens, Tres contained significantly more cells expressing OKT4 or Leu 3A antigens and cells bearing Fc mu receptors or containing Gall bodies. The Tsens fraction was enriched for cells with Fc gamma receptors but not for cells expressing OKT8 or LEU 2A. The results suggest that the Tres fraction is enriched for cells with putative helper markers and that the Tsens fraction is enriched for cells with some suppressor markers but also contains a large number of cells of monocytic lineage. However, the two fractions are not homogeneous with respect to Fc receptor status, presence of Gall bodies or antigens defined by OKT or Leu antisera, hence the use of theophylline sensitivity as a means of estimating the sizes of the helper and suppressor populations does not seem advisable.

Characteristics of suppressor factors generated from human mononuclear cells using concanavalin A and C-reactive protein.

Lymphocytes from normal human donors were cultured in Marbrook flasks in the presence of C-reactive protein (CRP), concanavalin A (Con A) or serum amyloid A protein low molecular weight component (SAAL) and the culture supernatants were tested for their ability to suppress the pokeweed mitogen (PWM) driven response of normal human lymphocytes. The supernatants from CRP and Con A stimulated lymphocytes, when added to cultures at initiation at a final concentration of 5%, suppressed the plaque forming cell (PFC) response in the three major immunoglobulin (Ig) classes. The suppressive effect could be removed by absorption of the culture supernatants with lentil lectin and with antisera to antigen specific helper and suppressor factors and could be recovered in the eluate. The results indicate that CRP stimulated cells exert their suppressive effect through a soluble mediator(s) which is similar to Con A generated suppressor factor(s) in respect to its time of action, its effect on the Ig classes M, G and A, its lack of cytoxicity and some structural aspects.

Spontaneous plaque forming cells in the peripheral blood of patients with systemic lupus erythematosus.

A reverse haemolytic plaque assay using staphylococcal protein A coupled to sheep red blood cells was set up in Cunningham chambers. Using this method, the numbers of Ficoll-Hypaque isolated peripheral blood lymphocytes (PBL) secreting IgG, IgA or IgM without preceding culture or mitogen stimulation were estimated in patients with systemic lupus erythematosus (SLE) and control subjects. Seven patients with clinically inactive SLE at the time of the study had values similar to those of the control subjects. In contrast, eight patients who had clinically active SLE had markedly increased numbers of PBL secreting IgG, IgA and IgM. Control experiments confirmed that the plaques were due to Ig secretion by lymphoid cells rather than to immune complexes adsorbed onto Fc receptor bearing cells or to passively adsorbed Ig. The results confirm the expected polyclonal B cell activation in patients with SLE and serial measurements showed that clinical relapses occurred only when the numbers of immunoglobulin secreting cells were high. Experiments in three patients with active SLE using native DNA prepared from T2 bacteriophage as the 'developing antigen' suggest that PBL secreting nDNA antibody can also be demonstrated by this method.

An audit of investigational services within general medical units.

Over a period of a year data was collected on the utilisation of investigational services by three general medical units. The pattern of practice was found to be very similar between the units. The average calculated daily test cost per patient ranged from +12-23 and the average dialy number of tests per patient was less than two. The services contributing most to costs were the relatively cheap but frequently-performed laboratory and radiological investigations. The implications of these findings in relation to hospital costs and to the education of medical staff is discussed.

A family study of leprosy: subcutaneous amyloid deposits and humoral immune responses.

One group of 11 aboriginal families, consisting of 27 persons with leprosy and 43 unaffected family members, and a second group of 26 patients with leprosy were studied in the Northern Territory of Australia. Amyloid deposits were sought in fine needle aspirates of subcutaneous fat and serological investigations relevant to amyloidosis and to the humoral immune response were done. The study showed unexpectedly high frequencies of amyloid deposits, evidence of persisting hepatitis B virus (HBV) infection, and antibodies to smooth muscle, to skin basement membrane, and to extractable nuclear antigens (ENA). Compared with unaffected family members, patients with leprosy had increased serum alpha-lipoprotein (alpha-LP) and were more often hepatitis B surface antigen (HBsAg) carriers but, contrary to expectations, the presence of amyloid, the alpha-LP level, serum amyloid associated (SAA) protein, and the HBsAg carrier state all appeared unrelated to the type of leprosy.

Liver disease and the e antigen in HBsAg carriers with chronic renal failure.

This study was undertaken to assess the frequency of development and the stages of evolution of chronic liver disease in patients with renal failure who are chronic carriers of hepatitis B surface antigen. Cirrhosis or chronic active hepatitis developed in five of 21 patients and could not be predicted by the initial histological appearance or by HLA-A and B typing but was associated with the e antigen in four of the five patients. However, the antigen was not a consistent indicator of a poor prognosis, as the four other e antigen positive patients did not develop chronic liver disease during the period of the study. Transmission of hepatitis B to spouses occurred in four cases, was fatal in one instance, and was associated with e antigen in three of the four. Determination of e antigen status in renal unit patients who are carriers of hepatitis B surface antigen may be of value to the patient and his home environment.

Immunological abnormalities in coeliac disease and their response to dietary restriction. I. Serum immunoglobulins, antibodies and complement.

Twenty-three patients with coeliac disease were studied whilst on a normal diet and again after a mean period of 15 months on a gluten-free diet. Serum levels of IgG, IgA and IgM, total haemolytic complement, C3, serum autoantibodies and precipitins to dietary proteins were compared to those in age and sex matched control subjects. There was considerable individual variation, but as a group, patients on a normal diet had significantly raised IgA and low IgM and an increased prevalence of antibody to reticulin, smooth muscle and dietary protein. These abnormalities disappeared during the period of dietary restriction suggesting that they are disease epiphenomena rather than primary pathogenetic factors.