Dipstick enzyme immunoassay for rinderpest antibody in cattle

A dipstick enzyme immunoassay (ELISA) has been standardized for the detection of rinderpest antibodies. One hundred and thirty bovine serum samples were analysed by the dipstick ELISA method and the results compared with the conventional plate ELISA method. The sensitivity was found to be similar in both methods. The dipstick ELISA does not require expensive micro-plates and an ELISA reader, and is recommended for use in field laboratories where the qualitative detection of rinderpest antibodies is required.     

Evaluation of nursing care in patients with myasthenia gravis

A retrospective study was made of 35 periods of hospital admission between January 1985 and September 1995, of 17 patients diagnosed in the Hospital Josep Trueta, Girona, as having myasthenia gravis. These were all the patients diagnosed as having myasthenia gravis in this ten year period. We studied the characteristics of their stay in hospital and evaluated the planning and practice of nursing care during these 35 periods. Nursing problems or diagnostic difficulties were found on 15 occasions, probably due to lack of knowledge of myasthenia gravis. When a patient had dysphagia, nursing care was increased and any problems identified. 29.4% of the patients were admitted to the intensive care unit. Since this disorder may be very serious, we consider that special training should be given to the nurses attending these patients. An understanding of this disorder and the possible treatments, complications, drugs, etc. would make it possible to give specific, individualized attention which would result in a higher standard of care.     

Detection of receptor mRNAs using nonradioactive in situ hybridization

To understand better the regulatory mechanism of cell function through bioactive substance such as hormones and cytokines, analysis of the gene expression of their receptors at the individual cell level is required. Since the presence of specific mRNA is a good index of gene expression, in situ hybridization (ISH) has been recognized as a powerful tool. Especially, nonradioactive ISH with synthetic oligodeoxynucleotide probe is a safe, quick and highly sensitive method. We have developed a unique method with thymine-thymine (T-T) dimer as a nonradioactive labeling, in which adjacent thymines are dimerized by ultraviolet light irradiation. The T-T dimers in hybrids are immunohistochemically recognized using an anti-T-T dimer antibody. In this article, we will present an example of ISH protocol, which works very well both T-T dimer and digoxigenin-labeled probes.     

Prognosis of myasthenia gravis: a retrospective study of 380 patients

The 9139 follow-up records of 438 myasthenia gravis (MG) patients were reviewed. Excluding those patients who were diagnosed 5 or more years after symptom onset (n = 37) and those who experienced only oculomotor symptoms throughout follow-up (n = 21), there were 380 patients. A survival analysis approach was used to assess the influence of prognostic factors on the following endpoints: (a) stable complete remission, (b) complete remission of at least 6 months and (c) pharmacological remission of at least 6 months. Early diagnosis was associated with a better prognosis with respect to all endpoints. Thymectomy also improved the prognosis but only for those patients without thymoma. Later MG onset was associated with a higher tendency to achieve pharmacological remission.     

Selective impairment of serum antibody repertoires toward muscle and thymus antigens in patients with seronegative and seropositive myasthenia gravis

We analyzed the antibody (Ab) repertoires of IgM and IgG of patients with seropositive and patients with seronegative myasthenia gravis (MG) toward self antigens by means of a quantitative immunoblotting technique using normal human tissue extracts as sources of self antigens. Repertoires of reactivities of IgG and IgM with liver, kidney and stomach antigens were conserved between myasthenic patients and controls. IgG and IgM Ab repertoires toward muscle antigens differed significantly between patients with seropositive MG and healthy donors, as assessed by multiparametric statistical analysis. Patterns of Ab reactivities to muscle antigens were similar in patients with seronegative MG and healthy controls. Antibody repertoires of IgG and IgM toward thymus antigens of both seropositive and seronegative MG patients, differed significantly from those of healthy individuals. Our results indicate that MG is characterized by a selective impairment of self-reactive Ab repertoires toward muscle and thymus antigens. The observation that self-reactive Ab repertoires toward thymus antigens are similar in patients with seropositive and seronegative MG suggests that both forms of MG share common immunopathological features.     

TCR-Vbeta usage in the thymus and blood of myasthenia gravis patients

In myasthenia gravis (MG) the muscle acetylcholine receptor (AChR) is the target of an autoimmune response. The anti-AChR response may originate in the thymus, which is abnormal in most MG patients and contains anti-AChR T and B cells. Microbial superantigens (sAg) may trigger autoimmune responses and in this study we sought clues as to whether sAg play a role in the pathogenesis of MG. We investigated the frequency of use of the different TCR Vbeta families by the thymus and blood T cells in MG patients and in control subjects, using a multi-primer PCR assay. Identical TCR-Vbeta usage was found in the thymi of MG patients and controls, except Vbeta2, which showed a small increase in MG patients' thymi. Blood T cells of MG patients used Vbeta4, Vbeta6, Vbeta15, Vbeta16 and Vbeta24 significantly more than those of the controls. Vbeta4 and Vbeta6 are the gene families most frequently used by anti-AChR CD4(+) cells in MG patients. Blood T cells from MG patients used Vbeta12, Vbeta14, Vbeta17 and Vbeta18 significantly less than controls. MG patients used Vbeta4 and Vbeta6 significantly more in the blood than in the thymus, while the opposite occurred for Vbeta7, Vbeta12 and Vbeta14. Controls used Vbeta17 more and Vbeta24 less in the blood than in the thymus. The preferential expansion of Vbeta4 and Vbeta6 in MG patients might reflect the immunodominance of certain AChR epitopes, or the action of a sAg outside the thymus. The minimal differences in the TCR-Vbeta usage in the blood and thymus of control subjects might be due to expansion of T cell clones specific for common antigens. Identical Vbeta usage in the thymi of MG patients and controls does not support an important role of the thymus as the location of anti-AChR sensitization when MG is clinically evident. The differences observed in the Vbeta usage in blood and thymi of MG patients are likely to be due to preferential Vbeta usage by the anti-AChR T cells in the blood.     

Detection of antibody IgG to HIV-1 in urine by ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant p24 as antigen for diagnosis of HIV-1 infection

Anti-HIV-1 IgG in urine was detected by an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant p24 gag protein (p24) of HIV-1 as antigen and beta-D-galactosidase from Escherichia coli as label. Anti-HIV-1 IgG in urine was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-recombinant p24 conjugate and recombinant p24-beta-D-galactosidase conjugate. The complex formed, consisting of the three components, was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG, eluted with epsilon N-2,4-dinitrophenyl-L-lysine, and transferred to polystyrene balls coated with affinity-purified (anti-human IgG gamma-chain) IgG. Bound beta-D-galactosidase activity was assayed by fluorometry. This assay was at least 3,000-fold more sensitive than conventional methods. The lowest signal among 49 asymptomatic carriers was 3.1-fold higher than the highest nonspecific signal among 100 seronegative subjects. The sensitivity and specificity were both 100%. The positivity could be confirmed by preincubation of urine samples with excess of the antigen. Thus, this assay would be a powerful tool for detecting IgG antibody to HIV-1 in urine.     

D-penicillamine-induced myasthenia gravis in a case of eosinophilic fasciitis

During the period 1983-1995, 200 chronic renal failure patients (115 males and 85 females) were parathyroidectomized for hyperparathyroidism in our Department. In all of them, the presenting clinical symptoms, physical signs, biochemical and radiological tests were typically those of hyperparathyroidism. One hundred ninety patients were operated for the first time whereas 10 were re-operated due to relapse of the disease; 3 of these cases were primary hyperparathyroidism, 182 secondary and 5 tertiary. All three primary hyperparathyroidism cases underwent removal of the adenoma; in the group of secondary hyperparathyroidism, 50 underwent removal of all the parathyroid glands found, 25 underwent total parathyroidectomy with forearm or deltoid autograft and 60 subtotal parathyroidectomy whereas in 39 and 8 patients only 3 and 2 parathyroid glands were found respectively. In the group of tertiary hyperparathyroidism, we removed only the hyperplastic gland detected as the operative detection of the rest was not possible. Ten cases were re-operated for removal of the remaining glands. No complications were noted postoperatively, apart from severe hypocalcemia in 20 cases, treated successfully by Calcium and Vitamin D administration. The highest relapse rate was noted among the 8 patients with only the 2 parathyroid glands removed. It seems that total or subtotal parathyroidectomy represents the most successful methods for surgical treatment of hyperparathyroidism complicating chronic renal failure.     

Thymus cells in myasthenia gravis selectively enhance production of anti-acetylcholine-receptor antibody by autologous blood lymphocytes

We investigated the role of the thymus in 16 patients with myasthenia gravis without thymoma by studying the production of anti-acetylcholine-receptor antibody by thymic and blood lymphocytes cultured alone or together. In 19 responders (with the highest receptor-antibody titers in their plasma), cultured thymic cells spontaneously produced measurable receptor antibody. Receptor-antibody production by autologous blood lymphocytes was enhanced by the addition of responders' thymic cells, irradiated to abrogate antibody production and suppression (P less than 0.01). This enhancement was greater and more consistent than that by pokeweed mitogen; it depended on viable thymic cells, appeared to be selective for receptor antibody, and correlated with the ratio of thymic helper (OKT4-positive or OKT4+) to suppressor (OKT8+) T cells (P less than 0.01). These results suggest that myasthenic thymus contains cell-bound acetylcholine-receptor-like material or specific T cells (or both) that can aid receptor-antibody production. This may be relevant to the benefits of thymectomy in myasthenia and to the breakdown in self-tolerance in this and other autoimmune diseases.     

Anti-idiotype monoclonal antibodies against anti-microcystin antibody and their use in enzyme immunoassay

Two different makes of bioimpedance spectrometer (UniQuest-SEAC SFB-3 and Xitron 4000B) were used for a series of measurements on volunteers and patients in intensive care. Although each machine was accurate over the frequency range 5 to 500 kHz when bench tested on model resistor-capacitor circuits, significant differences in their recorded impedance parameters appeared when used in vivo, especially on intensive care patients. A series of laboratory tests was performed on each machine simulating the situation in vivo to identify possible reasons for these differences. Whilst stray capacitance in the environment was identified as the major contributor to variability in high-frequency performance, interaction between electrode impedance and lead positioning was also a factor. The observed phase shift with frequency or time delay (Td) used in the Xitron modeling software appears to be the result of a time constant caused by stray capacitance and so is unlikely to have any biological meaning. Significant differences in the in vivo numerical values produced by bioimpedance spectrometers may be attributed to instrument design, data processing and, in particular, the clinical environment.     

Evaluation of a new enzyme immunoassay for Clostridium difficile toxin A

AIM: To evaluate a new enzyme immunoassay (EIA) method for detection of Clostridium difficile toxin by comparing it to cytotoxicity assay. To investigate the nature of false negative and false positive EIA results by evaluating clinical and therapeutic parameters. METHODS: 737 consecutive diarrhoeal specimens collected from patients clinically suspected of having C difficile colitis were tested for the presence of C difficile toxin by EIA for toxin A and by cytotoxicity assay. Clinical data were evaluated in all cases positive by either method. RESULTS: With the cytotoxicity assay as a gold standard, the specificity of EIA for toxin detection was 99.3% and the sensitivity was 62.2%. No false negative EIA specimens were obtained from patients already being treated for C difficile colitis. Among patients with cytotoxicity positive specimens, those with EIA positive samples had no clinical features distinguishing them from patients with EIA negative samples. CONCLUSIONS: Although specific, the new EIA method directed against toxin A lacks sensitivity compared to cytotoxicity. False negative EIA tests are not associated with concurrent treatment for C difficile colitis nor with any specific clinical features examined in our study.     

Incidence of SLE in a patient thymectomized for myasthenia gravis

The appearance of systemic lupus erythematosus (SLE) as well as thymopoietin (TP3) treatment in a patient who has undergone thymectomy because of myasthenia gravis (MG) is described. To the authors' knowledge this is the first case of this treatment upon the diagnoses above. The therapy was performed for 12 weeks by the Hungarian Thymotrinan (TP3) preparate according to a previous protocol worked out for Hodgkin's disease. At the beginning and during therapy, cellular and humoral immune parameters were monitored. Twenty months after the therapy the patient has remained in clinical remission for both diseases, MG and SLE, respectively. There is a brief survey of the biochemistry of thymic hormones as well as their clinical use, especially in the treatment of autoimmune disorders. The relative rarity of SLE after operative treatment of MG is under discussion together with the effectiveness of substitutional thymic hormone therapy.     

Detection of serum transforming growth factor-alpha in patients of primary epithelial ovarian cancers by enzyme immunoassay

OBJECTIVE: To survey the social outcome of patients with schizophrenia attending State mental health facilities in southern Tasmania. METHOD: Using the Statewide Mental Health Register, patients using inpatient and outpatient facilities who received a diagnosis of schizophrenia between 1981 and 1988 were identified (n = 771), and demographic and illness measures, and admissions and length of inpatient stay were compiled. The Life Skills Profile (LSP) was completed by mental health personnel for the 247 who were regular attenders or inpatients in 1991. RESULTS: Social morbidity as indexed by the LSP was highest in psychiatric hospital inpatients and patients in long-term rehabilitation programs, and lower in patients attending community centres. The majority of patients in suburban settings and attending community centres lived with their families, whereas patients in the inner city or in the rehabilitation service were mainly in hostel accommodation or living alone. Patients with schizophrenia attending State services were of a similar age range but had a longer duration of illness and more admissions, and had spent more days in hospital than patients who were not in regular contact with the service. CONCLUSIONS: The distribution of social morbidity in schizophrenia confirms that the public health system is supporting a group with high social morbidity. Patients with the highest morbidity are receiving the highest levels of care and intervention.     

Soluble adhesive molecules and cytokines in patients with myasthenia gravis treated with plasmapheresis

BACKGROUND: Plasma exchange (PE) is effective therapeutic method used in patients with myasthenia gravis (MG) refractory to common therapy and/or with life-threatening respiratory complications. Except from acetylcholine receptor antibodies (AChRAb) some other inflammatory mediators possibly activated in MG may be also removed during PE. METHODS AND RESULTS: Serum levels of soluble adhesion molecules (sICAM-1 and sVCAM-1), IL-6 and soluble receptors for IL-2 (sIL-2R), IL6 (sIL-6R) and TNF alpha (sTNF-R II) were measured in 20 patients (pts) with MG indicated to the treatment with PE. Pts were subdivided on the basis of the serum levels of AChRAb into 2 groups (8 pts with low AChRAb, 12 pts with high AChRAb). Soluble adhesion molecules and cytokines were measured before the 1st and last PE, at the end of the 1st PE and in the samples of plasma filtrate obtained during the 1st PE. Pts with MG had before the 1st PE higher serum levels of sICAM-1, sVCAM-1, sIL-2R and sTNF-R II than controls. Both the first PE and the course of PE led to the substantial decrease of serum levels of AChRAb, sICAM-1 and sVCAN-1, serum levels of sIL-2R and sTNF-R II were not, however, significantly influenced by both the single and the course of PE. There were high levels of AChRAb, soluble adhesion molecules and soluble cytokine receptors in plasma filtrate, too. Pts with high circulating AChRAb had higher serum levels of sICAM-1 and sVCAM-1 than pts with low AChRAb. CONCLUSIONS: Increased serum levels of soluble adhesion molecules and soluble cytokine receptors in pts with MG indicated to the treatment by PE suggest some systemic activation of immune response which is more pronounced in pts with high circulating AChRAb. PE led to the decrease of serum AChRAb and soluble adhesion molecules due to their effective filtration, but, on the other hand, serum levels of soluble cytokine receptors were not influenced by PE, in spite of their effective filtration which is probably counteracted by their increased production, possibly stimulated by the contact of the blood with synthetic membrane.     

Quantitative determination of serum anti ribosomal-P protein antibody by enzyme immunoassay

An enzyme immunoassay for serum anti-ribosomal P protein antibodies (anti-P) is developed, using highly purified synthetic ribosomal P peptides of the carboxyl terminal 22 amino acid sequence conjugated to human serum albumin (HSA) as an antigen. Anti-P levels were determined by subtracting the nonspecific binding activities to HSA. The concentration of anti-P which produced half of the maximal absorbance at 492 nm (OD492) given by saturating concentrations of anti-P in the ELISA plate was defined as 1 U/ml. The anti-P values in the samples were determined by referring to a standard curve made from a standard serum containing anti-P. Serum anti-P levels in 34 normal individuals were 5.52 +/- 8.39 U/ml (mean +/- SD). Anti-P in sera from 45 patients with systemic lupus erythematosus (SLE), 24 patients with rheumatoid arthritis (RA) and 27 patients with Beh?et's disease were also analyzed. The values for serum anti-P in SLE, RA and Beh?et's disease groups were 251.04 +/- 843.07 U/ml, 5.97 +/- 15.18 U/ml, and 2.62 +/- 3.35 U/ml (mean +/- SD) respectively. The positive ratio for serum anti-P in SLE patients was significantly higher than that in patients with RA or Beh?et's disease (p < 0.05 as determined by chi-square test). These results indicate that quantitative determination of serum anti-P by our enzyme immunoassay is a successful tool for the diagnosis of SLE.