The effect of ACTH treatment on lymphocyte subpopulations in patients with myasthenia gravis.

Blood lymphocytes were collected before, at various times during treatment and after treatment with ACTH in patients with myasthenia gravis. The relative proportions of thymus-derived (T cells) and bursa-derived (B cells) lymphocytes were determined. Eight of nine patients had a relative increase in B cells, whereas T cells showed a relative decrease. Myasthenic symptoms in these eight patients also were relieved. One patient who was not thymectomized showed virtually no relief in symptoms nor any changes in the relative T and B cell proportions or numbers, indicating a close association between the disease and the thymus-derived immune system.

Treatment of myasthenia gravis with anti-CD4 antibody: improvement correlates to decreased T-cell autoreactivity.

We treated a patient with severe myasthenia gravis with a chimeric (murine/human) anti-CD4 monoclonal antibody (cM-T412) for 7 days and followed the therapeutic effect by standardized muscle function tests, single-fiber electromyography, and immunologic examinations of disease-specific B- and T-cell functions. Clinical and electrophysiologic improvement began within 4 days, lasted for 3 months, and was maximal between days 16 and 58. The CD4+ lymphocytes decreased to a minimum of 80 cells per microliters of peripheral blood, recovered slowly during the first year of follow-up, and did not correlate with changes in disease severity. T-cell stimulation by human acetylcholine receptor was abolished by the treatment but became detectable at the time of worsening of symptoms. The concentration of acetylcholine receptor antibodies in serum was not decreased by the treatment. The results suggest that anti-CD4 antibody administration could be effective in the treatment of severe myasthenia gravis and indicate that acetylcholine receptor-specific T lymphocytes might contribute to the disturbed neuromuscular transmission in the disease.

Tumour necrosis factor-alpha polymorphism and secretion in myasthenia gravis.

The mechanism behind the association between MHC genes and myasthenia gravis (MG) is not fully understood. In the present study we studied the associations with polymorphisms at HLA-DR3, HLA-B8 and TNF-alpha genes in Swedish patients and healthy individuals. The TNF-alpha-308 allele 2 was associated with female patients having disease onset before the age 40 and with thymic hyperplasia. Analysis of strongest associations between MG and alleles close to TNF-alpha indicated that the association of TNF-alpha was possibly stronger than for HLA-DR3 and nearly the same as for HLA-B8. Peripheral blood mononuclear cells from patients positive for TNF-alpha -308 allele 2 had higher secretion of TNF-alpha when stimulated by anti-CD3 antibodies. Our results indicate that a subgroup of MG patients who have been previously shown to be associated with MHC genes may have a higher inducible TNF-alpha level in vivo, thus resulting the pathological changes in the thymus and the early onset of MG.

The effect of thymectomy on autoreactive T- and B-lymphocytes in myasthenia gravis.

Eleven patients with myasthenia gravis were followed for three years after thymectomy. Acetylcholine receptor-specific T-cell stimulation was found in 8/11 patients before operation as compared to 2/11 three years after thymectomy. Changes of T-cell antireceptor-reactivity were commonly paralleled by changes in disease severity. The numbers of cells secreting IL-2 upon stimulation with human acetylcholine receptor correlated with those secreting IFN-gamma. T-cell reactivity against a monoclonal acetylcholine receptor antibody did not decrease after thymectomy. Such reactivity could reflect a beneficial immune response counteracting anti-receptor reactivity. The frequency of autoantibody-secreting cells remained unchanged, while the serum concentration of acetylcholine receptor antibodies started to decrease one year after thymectomy. All examined thymus-cell suspensions contained autoreactive T- and B-lymphocytes. There was a preferential enrichment of autoreactive lymphocytes in the thymus in a few patients with recent onset of disease.

Polymorphisms at - 174 and in the 3' flanking region of interleukin-6 (IL-6) gene in patients with myasthenia gravis.

We examined the bi-allelic polymorphism at - 174 in the promoter region and the polymorphism in the 3' flanking AT rich region of the interleukin-6 (IL-6) gene in Swedish patients with myasthenia gravis (MG) and ethnically matched healthy individuals. There was no association between the polymorphisms and the disease. There was no relation of the polymorphisms to the clinical variables, the thymic histopathologies, the level of serum acetylcholine receptor antibodies or the concentrations of IgG and its subclasses. Our data yield no evidence for the IL-6 gene contributing to the disease susceptibility.

In vitro culture of human thymic epithelial cells in serum-free media.

Epithelial cells from human thymus were cultured in vitro at various serum concentrations and under defined serum-free conditions. A total of 238 cultures from 46 thymuses (MG and normal) were analyzed. Cells from fresh thymic tissue were explanted either as fragments or single cells after enzyme treatment. Serum-free as well as fetal calf serum (FCS) containing media based on Dulbecco's minimal essential medium and Ham's F-12 (DMEM/F-12) were found to be superior to MCDB 151 based serum-free media combinations, for the selective growth of thymic epithelial cells. In contrast, cultures based on RPMI 1640 medium supplemented with 1% FCS or more showed less epithelial cell selectivity and also supplement Ultroser G gave less fibroblast contamination. In serum-free media containing less than 0.1 mM ionic Ca, the cells had a smaller surface area and appeared more angular and also contained less keratin as compared to culture media with higher calcium contents. The development of serum-free conditions for in vitro growth of human thymic epithelial cells free of fibroblast contamination will facilitate studies of growth and maturation of the epithelial cells as well as investigations of their possible role in the development of myasthenia gravis.

Immunosuppressive drugs: azathioprine in the treatment of myasthenia gravis.

Azathioprine may be tried for treatment of severe myasthenia gravis, in a dose of 2 mg/kg BW. With careful monitoring the side reactions may be controlled. The therapeutic response will occur after a time delay of 4-8 months. About one-third of patients with type II MG (severe, late onset, HLA B8-) will proceed to a complete but azathioprine-dependent remission, and the remaining two-thirds to a marked improvement. In type I MG (early onset, HLA B8+) azathioprine is less effective but will help keep down the need for corticosteroids or plasmapheresis.

B-cell compartment in the thymus of patients with myasthenia gravis and control subjects.

An increased number of CD19, 20, and 22 positive B cells, compared to the number of Ig-positive cells, is regularly found in the thymic medulla of normal thymus, suggesting that a B-cell population normally resides in thymic medulla that lacks Ig expression. However, some of the CD20-positive cells seem to co-express keratin and MR19, suggesting an epithelial origin. The medullary B cells found in normal thymus could be precursors of the tumor cells in "mediastinal clear cell lymphomas of B-cell type." In follicular hyperplasia in MG, the medullary epithelial network is deformed and partly destroyed, and the interlobular/perivascular spaces are expanded. Follicles with follicular dendritic cells are found in both interlobular/perivascular spaces and "punched out" lesions in the medullary epithelium. The B cells are greatly increased in MG thymuses compared with control thymuses. These cells are found mainly in the follicles, but they are also dispersed in the medulla and the interlobular/perivascular spaces. The immunophenotype and distribution of B and T cells as well as the follicular dendritic cells in the hyperplastic follicles are similar to those of reactive follicles in lymph nodes. Our findings are consistent with the contention that in MG there is an autoimmune activation of B cells that normally reside in the thymic medulla. This activation leads initially to follicular hyperplasia in the medullary epithelium with destruction of medullary epithelial cells. The prolonged immune reaction in the autoimmune process induces a fibronectin-rich stroma formation and increased vascularization. The result is a remodeling of the thymic architecture with expansion of the perivascular/interlobular spaces replacing the destroyed medulla.

Effects of some immunosuppressive procedures on myasthenia gravis.

Clinical Observations. A total of 53 MG patients have been treated with different immunosuppressive methods (alone or combined) with the following effects: Thymectomy was performed in 38 patients. The improvement was excellent in 15, and moderate or uncertain in 20. In three patients severe long-lasting deterioration followed the operation. ACTH treatment (n=32): Initial deterioration during the 5-7 days of heavy ACTH treatment (1000 IU) was followed by an improvement lasting on an average 4 months. The improvement was good or moderate in 78% of the patients. Betamethazone treatment has been tried in six patients where ACTH and azathioprine was ineffective. In four of these patients the results were excellent. Azathioprine treatment has been given to 26 patients for periods up to 7 years. An improvement is measurable after 6-12 weeks and it seems maximal after about 1 year. Of the 26, 80% responded favorable with reduction in the need for cholinesterase inhibitors. Severe complications were seen in three patients with one death. Drainage of thoracic duct lymph was initiated in 14 patients up to 4 weeks with rapid improvement lasting as long as drainage was performed. Long-termed effects of the drainage may be present, however. Retransfusion of homologous cell-free lymph precipitated a return of the myasthenic symptoms. Biochemical Studies on Myasthenic Lymph. Using a membrane preparation from the electric organ from Torpedo marmorata and tritiated Naja naja siamensis neurotoxin we demonstrated a decreasing binding of toxin to the receptor in the presence of MG lymph gamma-globulin fraction. Gammaglobulins from controls showed almost no inhibition of the neurotoxin binding. Immunological Studies. An increased frequency of HL-A1 and 8 was found in female patients. LD typing was also performed. During a period of three weeks of thoracic duct drainage 130X10(9) or about 10% of total number of lymphocytes in the body were removed. In the lymph an initial decrease in the proportion of thymus-derived lymphocytes (T cells) occurred, which was accompanied by a sequent increase in the proportion of bone-marrow-derived lymphocytes (B cells). Towards the end of drainage this effect was reverted. Mitogenic stimulation using lymphocytes from thoracic duct drainage revealed no differences as compared to normal cells. The proportions of T and B cells was studied in the peripheral blood in nine patients treated with ACTH. During treatment there was an initial decrease in the proportion of T cells accompanied by a subsequent rise in the proportion of B cells, which was maximal after 3-10 days. These proportions were reverted to normal 1-5 days after the maximal change. The effect of azathioprine on T and B cells has also been studied.

Evaluation of a knowledge-based decision-support system for ventilator therapy management.

Evaluation of knowledge-based systems differs from that of conventional systems in terms of verification and validation techniques. Furthermore, evaluating medical decision-support systems is difficult because the field is thus far comparatively unexplored. This paper presents an evaluation of a medical knowledge-based system called VentEx that supports decision-making in the management of ventilator therapy. Real patient data from 1300 hours of patient care involving 12 patients with 6 diagnoses are used to validate the knowledge base. The results range from 4.5% to 15.6% disagreement between the setting recommendations produced by VentEx and a gold standard, and 22.2% disagreement for recommendations for weaning. A comparison between the standard and two physicians showed that VentEx produced advice of the same quality as the physicians.

Computer simulation of a patient end tidal CO2 controller system.

A computer model of the patient end tidal CO2 controller system has been developed and tested in simulation trials. It is intended to aid in finding the appropriate PI (proportional-integral) controller settings by means of computer simulation instead of real experiments with the system. The latter approach is costly, time consuming and sometimes impossible to perform. The simulator consists of two equations: the patient equation and the PI controller equation. The software has been written in the C language and can be run on an IBM-PC/XT. Some examples of the simulation trials, illustrating the choice of controller settings, are given.

Lung function analysis and optimization during artificial ventilation. A personal computer-based system.

In an intensive care unit a personal computer (PC) application for lung function analysis has been in use for 5 years. The PC system is applied to measure conventional and new parameters for diagnosis and therapy. The primary goal was to find parameters which could be used as optimization indices in optimal control systems for mechanical ventilation. Another clinical application of the PC system was as an automatic controller that stabilizes end-tidal CO2 concentration. The controller and the next application, the optimizer, could be integrated into an optimal control system. Such a system is described and a simulation trial of the integrated structure has demonstrated the potential.

Kave: a tool for knowledge acquisition to support artificial ventilation.

A decision support system for artificial ventilation is being developed. One of the fundamental goals for this system is the application of the system when a domain expert is not present. Such a system requires a rich knowledge base. The knowledge acquisition process is often considered to be the bottleneck in acquiring such a complete knowledge base. Since no single available method, for example interviewing domain experts, is sufficient for removing this bottleneck, we have chosen a combination of different methods. The different backgrounds of knowledge engineers and domain experts could cause communication restrictions and difficulties between them, e.g. they might not understand each others knowledge domain and this will affect formulation of the knowledge. To solve this problem we needed a tool which supports both the knowledge engineer and the domain expert already from the initial phase of developing the knowledge base. We have developed a knowledge acquisition system called KAVE to elicit knowledge from domain experts and storing it in the knowledge base. KAVE is based on a domain specific conceptual model which is a result of cooperation between knowledge engineers and domain experts during identification, design and structuring of knowledge for this domain. KAVE includes a patient simulator to help validate knowledge in the knowledge base and a knowledge editor to facilitate refinement and maintenance of the knowledge base.

Statistical models for prediction of arterial oxygen and carbon dioxide tensions during mechanical ventilation.

The possibility of constructing statistical models for prediction of alveolar oxygen and carbon dioxide tensions has been investigated in 20 mechanically ventilated patients in acute respiratory failure (ARF). Linear multiple regression analysis using PaCO2 and PaO2 as dependent variables was used to construct (a) models for individual patients, (b) models for specific diagnostic groups and (c) general models (all patients). The coefficient of determination (R2) was highest for the individual patient models (0.38-0.99) and lowest for the general models (0.28-0.49). In order to achieve a high predictive accuracy, models matching individual patients should be constructed on the basis of initial invasive blood gas measurement. Statistically derived models may bring better understanding of the behaviour of factors influencing arterial gas tensions in ARF and may be of value in the management of patients on mechanical ventilation.

Diagnostic utility of flumazenil in coma with suspected poisoning: a double blind, randomised controlled study.

OBJECTIVE: To assess the diagnostic value and safety of the benzodiazepine antagonist flumazenil in patients with coma of unclear origin with suspected poisoning. DESIGN: Double blind, placebo controlled, randomised study. SETTING: Intensive care unit at a major teaching hospital. PATIENTS: 105 Unconscious adults admitted consecutively with suspected drug overdosage during 18 months from a total of 362 cases of poisoning. Exclusion criteria were pregnancy, epilepsy, obvious poisoning with drugs identified unequivocally from information from relatives or others as other than benzodiazepines, and coma score greater than 10 on a scale graded from 4 to 20. Patients were allocated randomly to receive flumazenil (21 men and 32 women) or placebo (25 men and 27 women). INTERVENTIONS: Intravenous injection of flumazenil (10 ml, 0.1 mg/ml) or placebo (10 ml vehicle alone) given double blind over three minutes. MAIN OUTCOME MEASURES: Serum and urine concentrations of benzodiazepines, antidepressants, and several other agents; blood gas tensions; standardised evaluation on admission and five minutes after the injection by means of coma scale score and urgent diagnostic or therapeutic interventions indicated according to the history and clinical examination; standardised interview after the injection to try to ascertain further information; and adverse reactions. RESULTS: Benzodiazepines were found in the serum in 36 of the 53 patients in the flumazenil group and in 37 of the 52 who received placebo. The average coma scale score increased significantly after injection in the flumazenil group (6.4 v 12.1, p less than 0.001) but not in the placebo group. In the flumazenil group several interventions were rendered unnecessary by the injection: gastric lavage and urinary catheterisation (19 patients each), intubation (21), artificial ventilation and computed tomography of the brain (three patients each), blood culture and lumbar puncture (one patient each), and electroencephalography (two). In the placebo group the indications for these procedures did not change in any patient after injection. The 95% confidence interval for the difference in reduction of the frequency of indications for gastric lavage after injection between the two groups was 21% to 51%, that for intubation 25% to 55%, and that for urinary catheterisation 21% to 51%. In the flumazenil group 21 patients gave valuable information on their drug ingestion within 10 minutes after injection compared with only one in the placebo group (p less than 0.001). Nine adverse reactions were recorded in the flumazenil group, eight of which were graded as mild and one severe. The safety of the antagonist was acceptable, even though 60% of the patients in the flumazenil group had multiple drug poisoning including benzodiazepine. No epileptic seizures or arrhythmias were recorded. CONCLUSION: Flumazenil is a valuable and safe differential diagnostic tool in unclear cases of multiple drug poisoning.

VentEx: an on-line knowledge-based system to support ventilator management.

This paper will demonstrate the clinical application of a knowledge-based decision-support system called VentEx for ventilator management. VentEx has been implemented using a knowledge-based development tool on a PC under the Microsoft Windows multitasking environment. It is integrated into a computer aided ventilator system including the Siemens Elema Servo Ventilator 900 C equipped with a Servo Computer Module 990 and the CO2 analyser 930. The system provides advanced ventilator monitoring with expert advice concerning ventilator strategy and settings based on data from on-line monitoring. The knowledge base has been primarily validated and the system has been clinically tested by the intensive care unit staff. Different approaches such as knowledge acquisition, representation and system integration have been outlined and discussed.