HLA-patterns in patients with multiple sclerosis and type I diabetes mellitus: evidence for possible mutual exclusion of both diseases.

BACKGROUND: Type I diabetes mellitus (T1DM) and multiple sclerosis (MS), both immune-mediated diseases, rarely co-exist in the same individual or co-segregate in families. HLA susceptibility genes for T1DM (DRB1*0401, DRB1*0404, DQB1*0302, DRB1*0301, DQB1*0201) rarely occur in MS patients. HLA genes known to confer "resistance" to T1DM (DRB1*1501, DQB1*0602-DQA1*0102) predispose to MS. To test the hypothesis of mutually exclusive HLA patterns, patients affected by T1DM plus MS were compared to those of patients affected by either of the diseases alone in a case-control study. METHODS: Blood was sampled for analysis of HLA class I and class II alleles from 66 patients of German ancestry, of whom 33 had T1DM plus MS, and 33 had MS-only. For comparison to patients with T1 DM-only we referred to published data. HLA typing was performed using conventional serology (immuno-magnetic beads) and genotyping (SSP-PCR Dynal(R) SSP low/high resolution kits). RESULTS: Individuals with co-existing MS plus T1DM displayed the expected T1DM associated HLA-pattern (75.8% carried DRB1*04, 69.7% carried DQB1*0302, 42% were DR4, DR3 heterozygous), but failed to display the expected MS associated HLA-pattern (0% carried DQB1*0602, 3.1% carried DQA1*0102). The expected MS associated HLA-pattern of Caucasoid patients, however, was found in the MS-only patients (42% carried DRB1*1501-DQB1*0602, 58% carried DQA1*0102), while the prevalence of T1DM susceptibility and 'resistance' alleles was not different from the general population. The allele frequency of DRB1*1501 was 16/66, 24.2% in the 33 MS-only patients, and 0% in the 33 MS plus T1DM patients. The allele frequency of DQB1*0602 was 16/66, 24.2% in the 33 MS-only patients, and 0% in the 33 MS plus T1DM patients. The allele frequency of DQA1*0102 was 18/66, 27.3%, in the 33 MS-only patients, and 1/66 1.5% in the 33 MS plus T1DM patients. CONCLUSION: These data confirm the hypothesis of mutually exclusive HLA-patterns of T1DM and MS, and are consistent with a low rate of co-morbidity of both diseases.

Differential modulation of hippocampal signal transfer by tuberomammillary nucleus stimulation in freely moving rats dependent on behavioral state.

Tuberomammillary histamine neurons (TM) in the posterior hypothalamus project to extensive parts of the brain, including the hippocampal formation. The purpose of the present experiments was to investigate whether activation of the TM modulates signal transfer from the perforant pathway (PP) or ventral hippocampal commissure (VHC) to the dentate gyrus (DG) in freely moving rats. Paired pulses of electrical stimulation were delivered to PP or VHC, and evoked field potentials (fEPSPs and pop spikes) were recorded in the DG. Before activating PP or VHC, the TM was triggered by electrical stimulation. Experimentation was performed during four behavioral conditions: exploration, grooming, awake immobility, and slow-wave sleep. Electrical activation of the TM was found to modify dentate fEPSPs evoked by PP or VHC stimulation without generating a field potential by itself. Train stimulation of the TM (100 Hz, 500 ms) preceding paired pulses on the hippocampus by 50 ms decreased dentate fEPSPs in dependence of the ongoing behavior and the pathway stimulated. During exploration but not consummatory behavior, the PP signal was reduced when preceded by TM stimulation; during consummatory behavior but not exploration, the VHC signal was reduced. In contrast to other hippocampal afferents which increase pop spikes but leave fEPSPs unchanged, TM stimulation decreased dentate fEPSPs without affecting pop-spike activity. Thus, the TM-histaminergic system seems to modulate signal processing in the dentate gyrus in a specific way, exerting an inhibitory action on the entorhinal input only during learning-related exploratory behavior.

Tumor necrosis factor and interleukin-6 in critical illness polyneuromyopathy.

Critical illness polyneuromyopathy (CIPN) occurs in critically ill patients on artificial respiration. The pathophysiology of this disease is unknown. Because of the strong association with sepsis, the levels of cytokines, TNF and IL-6 were measured several times daily in patients having CIPN and in a control group of critically ill patients without CIPN. The diagnosis of CIPN was made on clinical criteria. Patients with CIPN had no significantly elevated levels of TNF or IL-6 as compared to controls.

Inhibitors of recombinant human erythropoietin in chronic renal failure.

This study investigates which factors influence the response of administered recombinant human erythropoietin (Re-HuEPO) with respect to the increase of haemoglobin in patients with end-stage renal disease. Pharmacokinetic parameters of administered Re-HuEPO in patients with end-stage renal disease and considerable differences in the amount of Re-HuEPO required ("Re-HuEPO-need") to obtain an increase of haemoglobin, revealed a pattern of dose-dependent first-order elimination without significant interindividual differences between the patients. As variable immunological inhibitors of erythropoietin are also absent, the administered Re-HuEPO seems to be equally available to the erythron in the various patients. In vitro incubation experiments with bone marrow cells show that the sera from patients with end-stage renal disease contain inhibitors of the erythropoietin-induced stimulation of bone marrow cells. As the patients' sera differ with regard to the degree of inhibition of erythropoietin bioactivity, this inhibition may also be responsible for the interindividual differences in amount of erythropoietin required. Besides a reduced endogenous production of erythropoietin, these inhibitors of the bioactivity of erythropoietin may also contribute to the pathogenesis of anaemia in patients with chronic renal failure.

Prostate specific antigen levels during and after external beam radiotherapy for localized carcinoma of the prostate: predictor of therapeutic efficacy.

For 105 patients with locoregional carcinoma of the prostate, prostate specific antigen (PSA) levels were evaluated before, during and after external beam radiotherapy. The median follow-up is 17 months. In 51 patients (48.5%) initial PSA levels exceeded the maximum normal value of 20 ng/ml. Nine patients kept non-declining high levels just after radiotherapy. Only one of these is free of disease. Assuming PSA levels decrease exponentially during radiotherapy, a mean half-life of 62 days (median 54, SD 26 days) was calculated. Three out of five patients with a PSA half-life of more than 88 days relapsed as compared to a 8% (3/37) relapse rate in patients with a "normal" half-life. Prolonged PSA half-life suggests residual disease. PSA levels are expected to further decrease after radiation. Six months after irradiation persistent high PSA levels were found in 14/51 (27.5%) patients. Only four of them had no evidence of manifest disease. Important negative prognostic factors for disease control in our series were non-declining high levels of PSA, a PSA serum half-life exceeding 88 days and persistence of elevated PSA values longer than six months after treatment. In our opinion, PSA is a valuable marker in the follow-up of prostate cancer patients during and after radiotherapy.

The spot test is not a reliable screening procedure for mucopolysaccharidoses.

To check the reliability of the Ames MPS paper spot test, which is based on the Azure A dye, we sent a series of urine samples to three laboratories where the spot test is part of the metabolic screening for mucopolysaccharidoses. In these laboratories false-negative results ranged between 19% and 35% and false-positive results ranged between 12% and 29% of all samples submitted. In contrast, the quantitative dimethylmethylene blue test (Clin Chem 1989;35:1472-7) detected an increased glycosaminoglycan content in all urine samples from mucopolysaccharidosis patients and gave no false-positive results. In the latter procedure, glycosaminoglycan content is expressed per millimole of creatinine, and age-dependent reference values are used. We conclude that the Ames spot test and other spot tests are unreliable as a screening procedure for mucopolysaccharidoses and should not be used to screen for these diseases.

Evaluation of a bench-top nephelometric immunoassay analyzer.

We evaluated the Technicon DPA-1 immunoassay analyzer on its analytical characteristics. Therefore we studied three assays: albumin in cerebrospinal fluid and IgG and transferrin in serum. When tested with the Cusum test for linearity albumin, IgG, and transferrin measurements showed no deviation from linearity. Closer examination revealed an abrupt difference of recovery (from 99 to 87%) in the albumin assay when the automatic dilution changed over from the primary analytical to the high analytical concentration range. One calibration was found sufficient for at least 14 days of measurement. Imprecision was well below the critical limits for reproducibility. We found reasonable agreement between the results from the DPA-1 and the results from comparison methods. However, the correlation plot of IgG showed lack of fit at a distinct segment of the regression line. This appeared to be caused by the poor recovery of the DPA-1 at the lower limit of the high analytical concentration range. The assays of IgG and transferrin were found insensitive for interference by hemoglobin, triglycerides, urea, and bilirubin. The albumin assay was found sensitive for bilirubin and triglycerides. No reagent- and sample-to-sample carry-over could be detected in the assays evaluated.

Uptake and concentration of steroid hormones in mammary tissues.

In order to exert their biological effects, steroid hormones must enter the cells of target tissues and after binding to specific receptor molecules must remain for a prolonged period of time in the nucleus. Therefore the endogenous levels and the subcellular distribution of estradiol, estrone, DHEAS, DHEA ad 5-Adiol were measured in normal breast tissues and in malignant and nonmalignant breast tumors from pre- and postmenopausal women. For estradiol the highest tissue levels were found in the malignant samples. No differences were seen in these levels between pre- and postmenopausal women despite the largely different peripheral blood levels. For estrone no differences were found between the tissues studied. Although the estradiol concentration was higher in the estradiol-receptor-positive than in the receptor-negative tumors, no correlation was calculated between the estradiol and the receptor consent. Striking differences were seen between the breast and uterine tissues for the total tissue concentration of estradiol, the ratio between estradiol and estrone, and the subcellular distribution of both estrogens. At similar receptor concentrations in the tissues these differences cannot easily be explained. Regarding the androgens, the tissue/plasma gradient was higher for DHEA than for 5-Adiol, and for DHEAS there was very probably a much lower tissue gradient. The highly significant correlation between the androgens suggests an intracellular metabolism of DHEAS to DHEA and 5-Adiol. Lower concentrations of DHEAS and DHEA were observed in the malignant tissues compared with the normal ones and the benign lesions. For 5-Adiol no differences were found and therefore these data do not support our original hypothesis on the role of this androgen in the etiology of breast abnormalities. Hence the way in which adrenal androgens express their influence on the breast cells remains unclear.

Purification and determination of the modifying protein responsible for the post-synthetic modification of creatine kinase (EC 2.7.3.2) and enolase (EC 4.2.1.11).

The purification of a serum protein, responsible for the postsynthetic modification of CK and enolase, is described. A purification of about 1300-fold could be reached after subsequent chromatography of human serum on DEAE cellulose and Sephacryl S-200 Superfine followed by affinity chromatography using antibodies against human serum albumin, C3 and C4 and against total human serum proteins. A recovery of 160% of modifying activity was found. The molecular mass and the isoelectric point of the modifying protein have been determined. It is concluded that the concentration of the modifying protein in human serum is less than 210 mg/l.

Endogenous concentration and subcellular distribution of estrogens in normal and malignant human breast tissue.

The endogenous concentrations and subcellular distribution of estrone and estradiol were measured in malignant and nonmalignant human breast tissue from pre- and postmenopausal women. The most striking finding was the significantly higher concentration of estradiol per g of tissue in the malignant tissues than in the nonmalignant tissues. The tissue concentrations of estradiol in pre- and postmenopausal women were similar despite the large differences in the peripheral plasma levels. No correlation was found between the estradiol receptor content and endogenous concentration and subcellular distribution of estradiol. No difference in the estrone tissue concentration was found between malignant and nonmalignant tissues. In comparison with human uterine tissues, which we have reported previously, human breast tissue "handles" estrogenic hormones differently from human uterine tissue. At equal concentrations of the estradiol receptor, concentrations and subcellular distribution of the estrogens are different in both tissues. It is concluded that the mechanism of action of estradiol via its receptor, a mechanism mainly based on studies in animal uterine tissue, applies only qualitatively to human breast cancer tissue.

Indices for the age of the creatine kinase M-chain in the blood.

The apparent activation energy of the CK reaction as well as the Michaelis-Menten constants and the isoelectric point of CK MM can be used as indices for the mean age of the CK M-chain in the blood in vivo and in vitro. Modifications in the CK M-chain take place in vivo in the blood and in vitro in a serum matrix. Gradual increases in the apparent activation energy are also observed both in vivo and in vitro. It is confirmed that the modification in the CK M-chain causes a rise in the apparent activation energy, mu. A gradual increase in apparent activation energy, due to the ageing process of the CK M-chain, was observed after myocardial infarction. A significantly increased value for u was observed at the time that total CK activity already had returned to reference values. In spite of the normal CK value, the apparent activation energy still indicated that there had been myocardial damage. The Michaelis-Menten constants for creatine phosphate and ADP are also influenced significantly by the modification in the M-chain. While the apparent activation energy increases, the Michaelis constants decrease in the order MM3, MM2, MM1. The Michaelis-Menten constants for both ADP and CrP can be used as an index for the mean age of the enzyme in the blood. The Michaelis-Menten constants for CrP and ADP show significant variations with the measuring temperature for virtually all CK MM forms.

Endogenous concentration and subcellular distribution of androgens in normal and malignant human breast tissue.

The endogenous concentrations and the subcellular distribution of dehydroepiandrosterone (DHEA) and 5-androstene-3 beta,17 beta-diol (ADIOL) were measured in malignant and nonmalignant human breast tissue from both pre- and postmenopausal women. DHEA 3-sulfate was measured only in the cytosol. A greater tissue-plasma gradient of DHEA was present with large variations. The highest concentration of DHEA and ADIOL occurred in the nuclear fraction (average, 2.9 and 1.6 times the concentration in cytosol). With respect to DHEA, this finding is remarkable because no specific binding protein in human breast tissue has been reported. The concentration of DHEA 3-sulfate was significantly higher in the cytosol of nonmalignant than in malignant breast tissues. No significant differences in tissue concentrations of DHEA and ADIOL were found in malignant and nonmalignant breast tissue. The concentration of estrogens was measured in the cytosol and the nuclear fraction of the same tissues, as reported in a previous paper. We found a significantly higher estradiol concentration in malignant tissue as compared to nonmalignant tissue. When the ratio of ADIOL to estradiol was calculated from the combined data, a significant difference was found only in the cytosol of premenopausal cancer patients versus normal women. No difference was seen in the postmenopausal women. No difference in the ADIOL:estradiol ratio was found between normal and malignant breast tissue of patients of the same menopausal status.

A study on post-synthetic modifications in alfa-alfa enolase (EC 4.2.1.11) brought about by a human serum protein.

Human alfa-alfa enolase is modified in the blood circulation by a serum protein for which the name 'modifying protein' is proposed. The protein occurs in every human serum tested and appears to be the same protein that is responsible for the post-synthetic modification in the M subunit of creatine kinase. Three alfa-alfa enolase forms, the original one plus two modified forms can be found in serum both in vivo and after in vitro incubation. The original alfa-alfa enolase is modified completely within a few hours in vitro in a pH-controlled human serum matrix at 37 degrees C. As the modification also takes place in vivo it is theoretically possible to acquire information about the activity of a disease process by doing one single determination of the amount of circulating alfa-alfa 3 enolase. A mechanism is proposed for the modification, where at first one of the two alfa chains is modified resulting in the alfa-alfa 2 form. Ultimately the second alfa chain is also modified. The alfa-alfa 1 form seems to be the completely modified alfa-alfa form. The three enolase forms differ in their isoelectric points but have similar Michaelis-Menten constants.

Measurement of endogenous subcellular concentration of steroids in tissue.

A reliable method for the extraction of steroid hormones from human uterine tissue and the subsequent measurement of these hormones in the subcellular compartments by radioimmunoassay is described. Extraction of radioactive steroid hormones from in vivo labelled human uterine tissue by different methods reveals that an almost quantitative extraction of steroid hormones from the nuclear fraction is obtained by sonication in ethanol-acetone. Extraction of steroid hormones with diethylether from a high speed cytosol is incomplete. Using a more potent denaturating agent prior to extraction with diethyl ether leads to complete extraction of unconjugated steroids.

Immunological measurements on the disappearance of creatine kinase MM from the circulation.

Both a two-site immunoradiometric assay and a two-site enzyme-linked immunosorbent assay for creatine kinase MM are described. Linearity, reproducibility and cross-reactivity of the assays are satisfactory. Creatine kinase MM incubated in a pH-controlled serum matrix loses its activity, but has its antigenic determinants affected as well. Of all the techniques used, only the immunoradiometric assay is capable of measuring part of the inactivated enzyme. Measurements with this assay on the sera of patients after a myocardial infarction show identical results for enzyme activity and creatine kinase protein quantity. The in vitro disappearance rate of creatine kinase activity is slow compared with the in vivo half-life of the enzyme. These two observations lead to the conclusion that creatine kinase is removed from the circulation long before it is inactivated in the blood stream.