Automated assay of vitamin B-12 by the Abbott IMx analyzer.

A nonisotopic assay of vitamin B-12 in human serum or plasma is described, performed with the Abbott IMx analyzer. The sample is first treated at pH > 12.5 to release bound vitamin B-12 and to convert all forms to cyanocobalamin. Next, the analyte is bound, at lower pH, by vitamin B-12-specific binding protein, immobilized to a solid phase of polymeric microspheres. Detection involves monitoring the activity of the tracer enzyme (alkaline phosphatase) coupled to a derivative of cyanocobalamin. Total assay precision is 7.9% for vitamin B-12 at 200 ng/L, 6.6% at 400 ng/L, and 6.7% at 800 ng/L. Assay sensitivity, calculated as 2 SD from the zero calibrator, is 37 (+/- 9) ng/L. The dynamic range extends to 2000 ng/L. Analytical recovery of 300 and 600 ng/L additions of vitamin B-12 to sera with basal concentrations of 30-400 ng/L was 102.5%. Results of the assay correlated well with those of commercially available radioisotope assays. No interference was observed in specimens from patients with pernicious anemia, chronic or acute myelogenous leukemia, or renal failure. Cross-reactivity with cobinamide (1 g/L) was < 0.00003%. Vitamin B-12 measurements for blood specimens drawn into serum, EDTA, or heparinized plasma-collection tubes agreed within 3%.


Transferrin index: an alternative method for calculating the iron saturation of transferrin.

We surveyed 140 clinical chemistry laboratories in Australia to establish which laboratory methods they used to determine serum iron status: 125 measured serum iron (Fe), 85 measured transferrin (TRF), 47 measured total iron-binding capacity (TIBC), and 14 measured both TRF and TIBC. Of the 55 laboratories routinely reporting TRF saturation (TS), 16 calculated TS directly as (Fe/TIBC) x 100, and 9 used [Fe/(TRF x 2)] x 100. Thirty laboratories measured TRF and converted it to an equivalent TIBC concentration; the derived TIBC was then used to calculate TS. We measured iron, TIBC, and TRF concentrations in 94 control subjects, 59 patients with alcoholic liver disease (ALD), and 20 with proven genetic hemochromatosis (GH). TS was compared with a transferrin index (TI = Fe/TRF) to determine whether both methods were sensitive for GH screening and which method gave the fewest false-positive results with discrimination limits of > 55% and > 1.0, respectively. All GH patients were detected by both TS and TI at these limits. One control subject had a TI > 1.0, whereas three control subjects had a TS > 55%. Nine patients with ALD had a TI > 1.0 and 11 ALD patients had a TS > 55%. Some iron-overload patients had lower than expected TS values compared with TI, possibly because of ferritin interference in the TIBC assay. Also, the precision of the TRF assay was better than that of the TIBC assay: CVs of 1.85-3.68% vs 6.17%. We therefore recommend that calculated TI replace TS in screening for iron overload.


Automated determination of fluvoxamine in plasma by column-switching high-performance liquid chromatography.

A column-switching system with high-performance liquid-chromatographic separation and ultraviolet detection is described for automated determination of fluvoxamine in human plasma or serum. Samples were injected and the drug was retained in a clean-up column [20 x 4.6 mm (i.d.)] filled with C8 reversed-phase material (10-micron particles). After unwanted material was washed out, the drug was eluted and separated with an analytical chromatography column, 4.6 x 250 mm (i.d.), filled with Nucleosil 100 CN (5-micron particles) with an acetonitrile:methanol:0.01 mol/L phosphate buffer eluent (188:578:235 by vol) at a flow rate of 1.5 mL/min for < 20 min and detected by spectrometry at 214 nm. With oxaprotiline as internal standard, fluvoxamine could be easily quantified, and it was well separated from endogenous plasma constituents and various psychoactive drugs. The detection limit was 10 micrograms/L (31.6 nmol/L), the analytical recoveries were 97-100%, and the relationship between drug concentration and detector response was linear from 0 to 1000 micrograms/L (3160 nmol/L). The automated method is suitable for therapeutic monitoring of fluvoxamine in the treatment of psychiatric patients.


Serotonin concentrations in plasma and variations during the menstrual cycle.

We used a specific and sensitive radioenzymatic method to establish a reference interval for the concentration of serotonin in platelet-poor plasma in 98 healthy volunteers (49 men, 49 women). The interval was 0-11 nmol/L with a median of 2.8 nmol/L. No difference in concentration in relation to sex or age was observed. In a group of eight very old volunteers (ages 86-92 years), however, concentrations were increased. In addition, we monitored the plasma concentrations of serotonin in 20 healthy women (ages 26-45 years) through two menstrual cycles. Periovulatory and premenstrual concentrations were greater than the serotonin concentration at the start of menstruation.


Nonimmunological assay of urinary albumin based on laser-induced fluorescence.

We describe the first nonimmunological assay of albumin in urine with a detection limit of 1 mg/L. The method is simple, rapid, and accurate. It is based on the probe Albumin Blue 670, which becomes highly fluorescent on binding to albumin. An inexpensive diode laser was used as the light source for measurement of laser-induced fluorescence. The assay was coupled to a flow-injection analysis system capable of running 20 samples per hour. The working range was 1-100 mg/L, which covered albumin concentrations found in nonpathological urine and in urine with slightly increased albumin. This range makes prediction of nephropathy possible at an early stage. Other serum proteins and hemoglobin do not interfere. The coefficients of variation were < 4% and < 7% within one day and from day to day, respectively. A correlation coefficient of 0.990 (n = 100) was obtained for comparison with the Behring nephelometric assay.


Laboratory evaluation of the Glucocard blood glucose test meter.

The Glucocard (Kyoto Daiichi Kagaku) blood glucose meter is designed for self-monitoring of blood glucose concentrations in capillary blood through use of an electrochemical test strip. Evaluated in this laboratory, the Glucocard had CVs of 4.6%, 6.6%, and 3.5% at blood glucose concentrations of 2.4, 4.1, and 18.9 mmol/L, respectively. The meter's response varied linearly with blood glucose concentration between 2.2 and 27.8 mmol/L. Hemolysis, urate, ascorbate, and acetaminophen interfered by > 5%. Different hematocrits, in the range 0.20-0.70, did not affect the measured glucose concentration. Comparison with glucose results measured in whole blood with a NOVA Stat Profile 5 instrument yielded the following: Glucocard = 0.898 NOVA--0.184 (r = 0.995). The main advantages of the Glucocard are its small sample volume (5 microL), wide linear range, and fully automated sample-handling steps, which reduce user-related variability.


Axon clinical chemistry analyzer evaluated according to ECCLS protocol.

We assessed the analytical performance of the Axon system (Bayer Diagnostici), according to the European Committee for Clinical Laboratory Standards guidelines, for assay of 12 analytes: cholesterol, creatinine, glucose, total protein, urea, uric acid, alkaline phosphatase, alpha-amylase, aspartate aminotransferase, creatine kinase, sodium, and potassium. The field evaluation lasted approximately 5 months and involved the collection of approximately 10,000 data points with the Axon. The following results were obtained: The highest CVs for controls and human sera at different concentration/activity values were 2.2% for within-run imprecision (n = 60; 3 days, pooled estimate) and 3.5% for the between-day imprecision (n = 20 days). Close correlation was found with results for patients' specimens assayed with comparative instruments (Hitachi 717 for substrates and enzymes, Beckman Synchron EL/E4A for electrolytes). No drift was observed during 8 h of operation. The linearity range was broad, sometimes exceeding the manufacturer's claims. No sample-, reagent-, or cuvette-related carryover was found. Measurement of control sera gave results within +/- 5% of the assigned values. We conclude that good reliability and practicability make the Axon system suitable for laboratories with various needs.


Two novel nonradioactive polymerase chain reaction-based assays of dried blood spots, genomic DNA, or whole cells for fast, reliable detection of Z and S mutations in the alpha 1-antitrypsin gene.

Two new nonradioactive polymerase chain reaction (PCR)-based assays for the Z and S mutations in the alpha 1-antitrypsin gene are presented. The assays take advantage of PCR-mediated mutagenesis, creating new diagnostic restriction enzyme sites for unambiguous discrimination between test samples from individuals who are normal, heterozygous, or homozygous for the mutations. We show that the two assays can be performed with purified genomic DNA as well as with boiled blood spots. The new assays were validated by parallel testing with a technique in which PCR is combined with allele-specific oligonucleotide (ASO) probes. In all cases tested the results obtained by the different techniques were in accordance. The new assays can be used for prenatal diagnostics and can be performed directly with boiled tissue samples. Because the new assays are easy to perform and reliable, we conclude that they are well suited for routine diagnosis.


Standardization with synthetic 22-kDa monomer human growth hormone reduces discrepancies between two monoclonal immunoradiometric assay kits.

Discrepancies among different methods for assaying human growth hormone have been described in various studies. The two major sources of discordant results are the heterogeneity of the antibodies and the different standardization bases used by the assay manufacturers. We propose standardizing assays with 22-kDa biosynthetic monomer human growth hormone diluted with the diluents supplied by the kit manufacturers. In a study of two monoclonal immunoradiometric assays (Hybritech, specific for the 22-kDa monomer; Sorin, recognizing also a 20-kDa variant hormone), standardization with 22-kDa monomer human growth hormone reduced by 63% the differences in results for 44 serum samples from children. The use of 22-kDa human growth hormone as a common standard, highly pure and easily available in large quantities, could help limit the interpretative problems in growth diagnostics.


Improving lactate analysis with the YSI 2300 GL: hemolyzing blood samples makes results comparable with those for deproteinized whole blood.

To obviate the well-documented problem of hematocrit dependency of the Yellow Springs Instruments (YSI) whole-blood lactate analyzer, we modified the dilution buffer by including a lysing reagent. This makes the results comparable with those of methods performed with deproteinized whole-blood samples. No centrifugation step is needed, thus preserving the convenience of the YSI instrument for stat and field use. The modification works equally well on plasma samples. Lactate concentrations measured in nonhemolyzed whole blood are not comparable with results for hemolyzed whole blood or protein-precipitated whole blood. We therefore recommend to all users of YSI equipment to lyse the erythrocytes before lactate determinations.


Cathepsin D concentration in breast cancer cytosols: correlation with disease-free interval and overall survival.

Cathepsin D (CD) is an aspartyl protease implicated in cancer metastasis. In this study of 331 patients, we show that patients with primary breast carcinomas containing high concentrations of CD have a significantly shorter disease-free interval (chi-square = 4.28, P < 0.05) and overall survival (chi-square = 7.7, P < 0.01) than patients with low concentrations. CD as a prognostic marker for overall survival was equally valuable for women younger (chi-square = 4.39, P < 0.05) and older (chi-square = 3.97, P < 0.05) than 50 years. CD was also a significant prognostic marker for overall survival within the estradiol receptor (ER)-positive subgroup of patients (chi-square = 5.79, P < 0.025), but not in the ER-negative subgroup. Patients with tumors containing high concentrations of CD and low concentrations of ER had shorter disease-free intervals (chi-square = 15.1, P < 0.001) and lower overall survival (chi-square = 20.9, P < 0.001) than patients with high concentrations of ER but low concentrations of CD.


Iohexol in serum determined by capillary electrophoresis.

Iohexol, a nonionic compound used as a contrast medium for angiography and as a measure of the glomerular filtration rate, was quantified in serum by capillary electrophoresis. Comparable results were obtained for serum samples deproteinized with acetonitrile or analyzed directly after 50-fold dilution with borate buffer. Serum samples were electrophoresed for 2.6 min at 12 kV in a borate buffer with detection at 254 nm and with 3-isobutyl-1-methylxanthine as internal standard. Acetonitrile deproteinization gave a greater sensitivity than did sample dilution. Between-run CVs were between 4.7% and 6.7%, and within-run CVs were between 2.5 and 3.2%. Analytical recoveries were 95-105%. Results of the method compared well with those by high-performance liquid chromatography (slope 0.96, intercept 0.005 g/L). This method demonstrates the potential of capillary electrophoresis for rapid and simple quantification of small molecules.


Fluorometric enzyme immunoassay of basic fibroblast growth factor with monoclonal antibodies.

We compared three different strategies for measuring basic fibroblast growth factor (bFGF) by fluorometric enzyme immunoassay (EIA). After optimizing conditions, we found that a primary anti-bFGF MAb directly conjugated with peroxidase gave the best detection limit for recombinant bFGF (approximately 30 ng/L, 3 pg/assay tube) in a two-site sandwich assay. The detection limit of methods based on biotinylated primary MAbs or on secondary antibodies followed by streptavidin-conjugated peroxidase was slightly lower than that of the above method. Using the most sensitive EIA examined in this study, we made a preliminary measurement of immunoreactive bFGF in sera of apparently healthy people and found it to be 190 (SD 32) ng/L (n = 48), in agreement with an earlier reported value (30-206 ng/L). Also, the concentration of immunoreactive bFGF in sera was above normal in 19 of 31 patients with stomach cancer.


In vitro effects of light on serum bilirubin subfractions measured by high-performance liquid chromatography: comparison with four routine methods.

The effects of light on serum bilirubin subfractions in vitro were investigated by HPLC and four routine methods for bilirubin analysis. By HPLC, the rate of photodegradation of unconjugated bilirubin (Bu) was nearly twice that of monoconjugated bilirubin (mBc) and threefold that of diconjugated bilirubin (dBc); delta bilirubin (Bd) was most stable against photoirradiation. In the diazo method, the rate of photodegradation of direct bilirubin was almost the same as that of the sum of mBc, dBc, and Bd determined by the HPLC method. However, the rate of photodegradation of indirect bilirubin was significantly lower (P < 0.001) than that obtained by HPLC, because approximately 30% of the bilirubin photoproducts reacted with the diazo reagent as indirect bilirubin. The rate of photodegradation of total bilirubin determined by the direct spectrometric method was lower than that determined by the diazo method, but equal to that of the total peak areas of HPLC. In the Ektachem method, bilirubin photoproducts affected total bilirubin negligibly, and Bc and Bu positively, so that the value of Bd decreased. In the bilirubin oxidase method, bilirubin photoproducts were oxidized enzymatically by both the total and direct bilirubin reagents. We re-emphasize the importance of shielding serum from light to avoid generating bilirubin photoproducts that interfere with the accurate determination of serum bilirubin subfractions. We also recommend HPLC analysis as a standard method for bilirubin measurement.


Superiority of dynamic over static reference intervals for intact, midmolecule, and C-terminal parathyrin in evaluating calcemic disorders.

We compared the clinical performance of assays of intact, C-terminal, and midmolecule parathyrin (PTH), when used with either a dynamic reference interval (based on the range of serum PTH concentrations observed in 35 healthy individuals during acute modifications of their blood calcium concentrations) or a gaussian (mean +/- 2 SD) interval derived from normocalcemic individuals. Dynamic intervals were substantially different from gaussian intervals, with half of the area delimited by the gaussian limits for calcium and intact PTH concentrations, and one-third for both C-terminal and mid-PTH assays, being outside the range of values observed during the dynamic tests. Use of the dynamic intervals increased the average clinical sensitivity of the three assays for detecting primary hyper- and hypoparathyroidism from 68% to 97% (and up to 100% for the intact and C-PTH assays). Even though only the intact PTH assay allowed complete separation between primary hyperparathyroid and nonparathyroidal hypercalcemic patients, the average proportion of patients correctly classified in this latter category was increased from 40% to 70% by the use of dynamic intervals. We conclude that gaussian reference intervals are largely responsible for the poor clinical sensitivity of many types of PTH immunoassays and that they should be replaced by dynamic reference intervals when evaluating calcemic disorders.


Concentrations and molecular forms of C-type natriuretic peptide in brain and cerebrospinal fluid.

To develop a radioimmunoassay (RIA) specific for human C-type natriuretic peptide (hCNP), we used a highly specific antiserum raised in rabbits. Quantitative inhibition tests with various natriuretic peptides demonstrated that the 50% inhibitory dose of hCNP was 15 fmol, whereas those of other natriuretic peptides were 10(5)-fold higher, indicating a specificity satisfactory for determining concentrations of hCNP in tissues. Using this antiserum, we detected immunoreactive hCNP (ir-hCNP) in various regions of human brain and spinal cord, as well as in cerebrospinal fluid (CSF). The ir-hCNP concentrations in human neural tissues were approximately 10-fold higher than those of immunoreactive human atrial natriuretic peptide (ir-hANP). The mean (+/- SD) concentration of ir-hCNP (72.0 +/- 17.8 ng/L) in CSF also was 10-fold higher than that of ir-hANP (5.2 +/- 2.1 ng/L). Using gel-permeation chromatography, we identified two molecular forms of ir-hCNP in brain and CSF: a 2-kDa form corresponding to mature hCNP, which is composed of 22 amino acid residues (hCNP-22), and a 5- to 6-kDa form corresponding to an N-terminally extended molecule (hCNP-53). The latter form was predominant in brain; the former was the main constituent of hCNP in CSF. These results support the hypothesis that hCNP is a major natriuretic peptide, is synthesized in human brain, and functions in human central nervous tissues.


Increased prostaglandin PGE2 and 6-keto-PGF1 alpha immunoactivity in incubates of gastric mucosa and forestomach of nephrectomized rats.

Prostaglandin E2 (PGE2)- and 6-keto-PGF1 alpha-like immunoactivity was measured in incubates of forestomach and gastric corpus mucosa in (a) unoperated rats, (b) rats with sham-operation of the kidneys and (c) rats with bilateral nephrectomy. In addition the mean ulcer area in the forestomach and gastric mucosa was assayed in all three groups of rats. The PGE2- and 6-keto-PGF1 alpha-like immunoactivity in gastric mucosa incubates and 6-keto-PGF1 alpha in the forestomach incubates was almost the same in unoperated and sham-operated rats. Increased 6-keto-PGF1 alpha in the forestomach and of PGE2 and 6-keto-PGF1 alpha in the gastric mucosa was found in rats with bilateral nephrectomy before gastric lesions were seen. A higher mean ulcer area occurred in the forestomach and gastric mucosa 24-48 h after bilateral nephrectomy. We conclude, that the increased PGE2- and 6-keto-PGF1 alpha production by the gastric mucosa and forestomach was associated with the loss of normal renal tissue function. Despite the protection of PGE2 and PGI2 by gastric tissue, gastric lesions nevertheless occur in acute uraemic rats.


Routine use of hair root or buccal swab specimens for PCR analysis: advantages over using blood.

We report the use of hair roots and buccal cells as specimens of choice for DNA analysis of genetic diseases in a service laboratory. Our protocols using these specimen types show superiority to those using blood specimens in the areas of collection, transport, storage and overall cost. Our experience using these specimen types for 319 cystic fibrosis delta F508 mutation tests and 62 Leber's hereditary optic neuroretinopathy mutation tests leads us to recommend that hair roots and buccal cells should be evaluated as specimens of first choice when developing PCR DNA analysis.


Determination of enterokinase activity by measuring the disappearance of trypsinogen.

An electrophoretic technique for the determination of enterokinase activity is described. The natural substrate trypsinogen is hydrolysed in the presence of soybean trypsin inhibitor. Under the conditions of assay, neither the trypsin inhibitor nor the trypsin-trypsin inhibitor complex are detected. Enterokinase activity can be determined in biological materials such as duodenal aspirates without any interference from trypsin activity. A significant correlation exists between the present technique and a spectrophotometric technique by which the liberation of trypsin activity is used to determine enterokinase activity.


A flow-through system for the determination of salicylate in blood.

A flow-through system for the determination of salicylate in blood is described. It consists of a Clark-type oxygen probe with the enzyme salicylate hydroxylase chemically bound to polymer supports. This probe, when inserted into a flow-through cell in the presence of salicylate, oxygen and NAD(P)H, produced a current signal proportional to the concentration of salicylate. Salicylate was assayed in the range 10 mumol/l up to 200 mumol/l with a detection limit of 3 mumol/l. Salicylate analysis was optimized by selecting the appropriate pH, buffer, temperature, enzyme immobilization and NAD(P)H concentration. The accuracy of this method was evaluated by recovery studies on 15 sera. Analysis of salicylate in serum sample was performed by diluting samples thirty fold with phosphate buffer to fit the calibration graphs. The response time of the probe was 2 min and 6 min was the time to perform a single analysis. Results compared with the TDx procedure correlated well.


Purification and characterisation of antigenic gliadins in coeliac disease.

Two gliadins, known to be especially antigenic in coeliac disease, were purified to homogeneity by a series of ion-exchange chromatography steps. Their N-terminal amino acid sequences showed minor differences but clearly classified them as gamma-type gliadins. The purified gliadins were further characterised with respect to amino acid composition, molecular mass and E1(1%)cm at 276 nm. Based on these properties it is suggested that one of them is identical to a gamma-type gliadin, earlier characterised by its nucleotide sequence, whereas the other has not previously been described. The purification procedure may form the basis for the development of a more differentiated analysis of circulating antibodies for diagnosis and makes clinical testing of the toxicity of defined gliadin peptides feasible.


Human urinary protein 1: evidence for identity with the Clara cell protein and occurrence in respiratory tract and urogenital secretions.

Protein 1 (P1), a low mol mass urinary protein of unknown function, has been purified, sequenced and quantified in human biological fluids. The molecular size, subunit composition and partial amino acid sequence of P1 are similar to those of the 10 kDa Clara cell protein (CC10), a lung secretory protein. P1 is found in high concentrations in sputum, bronchoalveolar lavages, urine and semen of healthy individuals and in urine of some pregnant women. Contrary to what is claimed, P1 or CC10 is not a specific and unique product of the lung, but like its homologue in rabbits (uteroglobulin) it is also present in urogenital secretions. P1 or CC10 may act as a natural immunosuppressor protecting the respiratory and urogenital tracts from unwanted inflammatory reactions.


Enzyme induction by drugs and toxins.

Enzyme induction by drugs mostly concerns those enzymes involved in drug metabolism: cytochromes P-450, UDP-glucuronosyltransferases, glutathione S-transferases, gamma-glutamyltransferases and epoxide hydrolases. A large variety of molecular forms exists, but not all of them are inducible (e.g. the inducible cytochromes P-450 in man are members of family IA, IIA, IIC, IIE, IIIA). Induction is most common in the liver, but also occurs in other organs (lung, placenta, lymphocytes). Over the past 20 years a relatively small number of drugs and environmental chemicals have been identified as enzyme inducers, perhaps fewer than early studies suggested. Information on inducing properties must be obtained as early as possible during the development of a new drug and made available to clinicians and clinical chemists when the drug is marketed. The main consequences of enzyme induction are changes in pharmacokinetics of the drug itself or of an associated drug. Much progress has been made in methods to identify these inducers.


Alkaline phosphatase as a reporter of cancerous transformation.

Serum levels of alkaline phosphatase (AP) have been used in the clinical evaluation of numerous diseases, including malignancies, for half a century. The aberrant expression of AP genes in cancer cells has led to the suggestion that APs are oncofetal proteins and thus, could be involved in tumorigenesis. Tumors which express these AP isozymes can be broadly divided into two groups: (a) those with an enhanced production of an isozyme normally expressed in the tissue (eutopic expression) and (b) those showing expression of one or more isozymes not identified in the normal tissue (ectopic expression). Moreover, many tumors show simultaneous expression of two or more different AP isozymes. In the absence of known biological functions of the AP isozymes several different mechanisms underlying their expression in tumor cells could explain the findings. In an attempt to clarify the function of APs, this laboratory is engaged in the study of unique properties of the mammalian APs as possible clues to their function, i.e., (a) the phosphatidylinositol glycan attachment of APs to the cytoplasmic membrane, (b) the uncompetitive inhibition properties of APs and (c) the extracellular matrix binding domain of APs. This laboratory is also undertaking the task of targeting each of the mouse AP isozymes by homologous recombination to generate mouse models of hypophosphatasia to analyze in detail the in vivo consequences of AP isozyme deficiencies.


Changes in enzyme expression related to differentiation and regulatory factors: the acid phosphatase of osteoclasts and other macrophages.

Human tartrate-resistant Type 5 acid phosphatase is a unique isoenzyme encoded by a gene located on chromosome 19. It is a member of a widely-distributed and structurally highly-conserved group of iron-containing proteins. It is normally expressed in certain tissue macrophages, notably osteoclasts and alveolar macrophages, but is virtually absent from the precursor monocytes. Factors which enhance or inhibit expression of this specific isoenzyme can be studied in monocytes and osteoclasts cultured in vitro. This provides opportunities to develop the use of Type 5 acid phosphatase as a reporter of pathophysiological events and an essential, though not sufficient, role in bone resorption by osteoclasts has been established by such studies.


Plasmodium falciparum induced perturbations of the erythrocyte antioxidant system.

Erythrocyte antioxidants catalase, superoxide dismutase, reduced glutathione and glutathione peroxidase were studied in cells harbouring different growth stages of Plasmodium falciparum. Catalase and superoxide dismutase showed significant decrease during parasite maturation indicating hampered metabolism of hydrogen peroxide and superoxide anions. Glutathione peroxidase also exhibited a downward trend during the growth of P. falciparum, while there was a moderate accumulation of reduced glutathione. These findings suggest decreased utilization of the reduction potential in detoxification of reactive oxygen species. The fall in all three antioxidant enzymes studied was highly significant (P less than 0.001) in erythrocytes with mature stages of the parasite (trophozoites, schizonts). The increased vulnerability of erythrocytes to damage, which parallels the growth phases of the parasite emphasizes the need for early treatment of P. falciparum malaria to minimise red cell destruction and the resulting anaemia.


Use of an azo derivative immobilized on an anionic resin for the determination of aluminium in water and dialysis fluids.

We studied the retention of aluminium on Chromotrope-2R immobilized on Amberlite IRA-400 resin with a view to the subsequent atomic spectrometric determination of the metal. The determinative method thus developed allows the determination of aluminium in water and dialysis fluids over the concentration ranges 0.92-19.46 and 0.37-0.74 mumol/l, respectively.


Investigation of urea cycle enzyme disorders by 1H-NMR spectroscopy.

High resolution proton nuclear magnetic resonance spectroscopy (1H-NMR) has been used to study patients with inborn errors of the urea cycle to evaluate further the diagnostic potential of this technique. The 1H-NMR metabolic profile from the urine of patients with citrullinaemia and argininosuccinic aciduria consistently demonstrated the presence of the diagnostic metabolites citrulline, N-acetylcitrulline and argininosuccinate, respectively. The profile from the urine of patients with ornithine carbamoyl transferase deficiency, is potentially diagnostic, but orotate was only detected in samples from three out of four patients. The characteristic fingerprint that each of the metabolites produces is unlike that of any other we have seen, including analogues of the metabolites which are structurally very similar such as arginine, ornithine and aspartate. The level of excretion of the metabolites from the patients with citrullinaemia and argininosuccinic aciduria has been well within the range of NMR detection.


Diagnosis of medium chain acyl CoA dehydrogenase deficiency by measurement of cis-4-decenoic acid in dried blood spots.

Using gas chromatography-mass spectrometry (GC-MS), with selected ion monitoring, a method for measurement of cis-4-decenoate in dried blood spots was developed. Using this assay, the concentration of cis-4-decenoate was determined in blood spots taken from a control population, seven children with medium chain acyl CoA dehydrogenase (MCAD) deficiency who were well at the time of sample collection and an asymptomatic sibling of a child with MCAD deficiency. cis-4-Decenoate was elevated, above the control range, in all the children with MCAD deficiency and in the previously undiagnosed sibling. It is concluded that measurement of cis-4-decenoate in dried blood spots provides a reliable and sensitive test for MCAD deficiency, that could be used in screening programmes.


Clinical test of renal guanidinoacetic acid metabolism by oral citrulline and creatine loading.

We devised a clinical test of renal metabolism based on the synthesis of guanidinoacetic acid from citrulline in the proximal convoluted tubule. Intravenous administration of a citrulline/creatine solution to rats with modified levels of renal glycine amidinotransferase activity revealed a strong correlation (r = 0.921) between this activity and urinary guanidinoacetic acid excretion. Citrulline (1.75 g) and creatine (1.50 g) were administered orally to healthy individuals and patients with chronic glomerulonephritis. In the healthy individuals, urinary guanidinoacetic acid excretion increased 5-fold by 2 h after dosing (15.1 +/- 2.2 vs. 2.8 +/- 1.1 mg/h). In the glomerulonephritis patients, blood clearance of citrulline decreased as the creatinine clearance decreased and urinary guanidinoacetic acid excretion also decreased. Of 56 patients with glomerulonephritis or diabetes mellitus, one had increased urinary guanidinoacetic acid excretion associated with an excess of adrenal androgens. This test appears a useful, noninvasive and simple method for examining the metabolic activity of the renal proximal convoluted tubules.


Diagnostic applications of repetitive DNA sequences.

The potential, the advantages and the different areas of diagnostic applications are discussed for the various categories of repetitive DNA sequences. Since all eukaryotes are characterized by genomic redundancy, these sensitive, rapid and comparatively simple techniques are revolutionizing many a field of clinical and experimental diagnostics.


Alterations of purine metabolism in mononuclear cell populations of first degree relatives of insulin-dependent diabetic individuals with disturbed glucose tolerance.

In a T lymphocyte and macrophage-depleted mononuclear cell population of the peripheral venous blood of 10 of 41 first degree relatives of insulin-dependent diabetic individuals who had or had had disturbed glucose tolerance adenine uptake rates were significantly increased, the relative adenine incorporation rates into the adenine nucleotides, however, were diminished. Values were compared with those of 30 controls. In 7 of 9 investigated individuals with increased adenine uptake rates antibody-dependent cellular cytotoxicity against rat Langerhans islets (ADCC) was increased in the same cell population. In these individuals the number of diabetes manifestations was relatively high. Adenine uptake rates, ADCC and glucose tolerance changed with time.


Melatonin and 6-sulfatoxymelatonin circadian rhythms in serum and urine of primary prostate cancer patients: evidence for reduced pineal activity and relevance of urinary determinations.

The circadian rhythms of melatonin and 6-sulfatoxymelatonin (aMT6s) were analyzed in serum and urine of young men (YM, n = 8), of elderly patients with benign prostatic hyperplasia (BPH, n = 7) and of patients of similar age with primary prostate cancer (PC, n = 9). The data expressed as concentration and in urine also as hourly excreted quantity were analyzed chronobiologically by the single cosinor method and, subsequently submitted to linear regression analyses. Circadian rhythms were detected in all cases except for the excreted quantity of melatonin. The circadian patterns of melatonin and aMT6s in serum were very similar in the different groups and regression analyses showed close correlations between both variables. MESOR and amplitude were significantly depressed in PC (40-60%) as compared to BPH and YM indicating that the depression of serum melatonin in PC is due to a reduced pineal activity and is not caused by an enhanced metabolic degradation in the liver. Acrophases of serum melatonin occurred between 01:34 and 03:26 h and of serum aMT6s between 03:58 and 04:35 h. Circadian rhythms similar to those of serum melatonin and aMT6s were found in urine, particularly for aMT6s excretion as well as melatonin concentration; the determination of both parameters in overnight urine samples closely correlated with the nocturnal peak of circulating melatonin. These results imply that it is feasible to estimate changes in pineal function of prostate cancer patients by means of non-invasive determination using urinary melatonin and aMT6s.


Lipid and apolipoprotein changes after orthotopic liver transplantation for end-stage liver diseases.

Orthotopic liver transplantation was performed in 37 patients with different endstage liver diseases. Changes in lipid and apolipoprotein concentrations were followed daily from day 1 to 20 after surgery and regularly thereafter until 12 months. When the acute effects of surgery had cleared away, there was a sharp drop in HDL-C, apo A-I and A-II from day 1 to 5, a stabilization at their lowest values from day 5 to 15 and then a progressive rise. Contrasting with this drop, triglycerides, apo B, C-II and C-III increased from day 1 to 5 with variable concentrations thereafter. Apo SAA considerably increased early after surgery and remained significantly higher than normal in most patients after 12 months. All other parameters returned to normal from 3 to 6 months after transplant. The mechanism leading to these lipid and apolipoprotein changes are discussed with respect to the distant effect of infusions, re-alimentation, immunosuppressive therapy and lipoprotein metabolism. The apolipoprotein concentrations appear very useful indicators of functional liver recovery.


Relationships between androgen and estrogen sulfates in breast cyst fluid.

In 28 breast cyst fluids obtained from 20 patients (age 29-65 years) sodium, potassium and the sulfates (S) of estrone (E1), estradiol (E2), dehydroepiandrosterone (DHEA) and androsterone (A) were determined. The radioimmunoassays (RIA) used were validated for this particular biological fluid. According to electrolyte ratio (Na+/K+) the cyst fluids were subdivided into two groups: the first with low (less than 3) (n = 16) and the other with high (greater than 3) (n = 12) values. Markedly higher steroid sulfate levels were observed in the first group, the mean levels being: 147.7 nmol/l, 54.6 nmol/l, 108.1 mumol/l and 158.0 mumol/l for E1S, E2S, DHEAS and AS respectively. The mean levels in the second group were: 13.6 nmol/l, 6.7 nmol/l, 68.8 mumol/l and 33.6 mumol/l for E1S, E2S, DHEAS and AS, respectively. In the first group only E1S and E2S levels were significantly correlated (r = 0.51; P less than 0.05). Conversely, the steroid sulfate levels were significantly correlated with each other in the group with high electrolyte ratio. These data have confirmed preceding results and have clearly shown that breast cyst fluids with low electrolyte ratio contain more E2S than the other group. This finding might be correlated with the fact that patients with these breast cysts lined by with apocrine epithelium may be at a greater risk of breast cancer than those with the other type.


Increased membrane activity of glyceraldehyde 3-phosphate dehydrogenase in erythrocytes of patients with homozygous sickle cell anaemia.

Membrane-bound glyceraldehyde 3-phosphate dehydrogenase activity was measured in erythrocytes from 43 patients with sickle cell anaemia, 24 heterozygous and 27 controls. A significant increase of the activity was found among the patients but no such difference was observed between the heterozygous and the control individuals. The patients showed nearly non Gaussian distribution of the enzyme activity and were subgrouped on the basis of these results, subgroup I had normal values and subgroup II showed markedly increased activities. The patients in subgroup II had significantly lower blood haemoglobin concentrations and significantly higher lactate dehydrogenase activities in serum than subgroup I. The subgroups did not differ in blood reticulocyte counts, serum bilirubin, serum iron concentrations or band 3 protein content of erythrocyte membranes.


Pattern visual evoked potential luminance and multiple sclerosis.

Pattern visual evoked potentials (PVEPs) were recorded from 111 patients classified as having possible, probable or definite multiple sclerosis. Patients were stimulated with a checkerboard pattern using high and low luminances in order to test the hypothesis that an attenuated pattern luminance increases the detection rate of PVEP abnormalities. With increasing certainty of diagnosis, there was a concomitant increase in the incidence of PVEP abnormalities. However, there was no evidence that stimulating with a lower luminance pattern enhanced the sensitivity of the test. The same findings were also apparent when the patient data was analyzed according to the presence or absence of a history of optic neuritis or other visual symptoms. It is concluded that, within the luminance limits used in this study, the role of varied luminance in detecting demyelinating lesions in the optic nerves using the PVEP is minimal, although there was some limited evidence that a high level of luminance may be more appropriate than a low level.


Differences in pattern visual evoked potential (PVEP) between hemodialysis and peritoneal dialysis patients.

The visual evoked potential was recorded in peritoneal and hemodialysis patients as compared to normal controls. By using the appropriate visual stimulus we were able to disclose specific VEP abnormalities for each of the two dialysis groups. The dissociation found between the latency of N70 and P100 in peritoneal dialysis patients suggests a possible postsynaptic visual abnormality not described previously. The correlation between the high serum aluminum and the P100 latency of peritoneal dialysis patients requires further investigation.


Alternating periodic lateralized epileptiform discharges (cerebral bigeminy).

Periodic lateralized epileptiform discharges (PLEDs) are seen, as a rule, following acute brain damage. We presented a 54-year-old diabetic male with ischemic coronary disease. Following cardiopulmonary arrest, the patient had a particular EEG pattern of PLEDs, characterized by two recurrent discharges. To our knowledge, this is the fourth published case of such EEG alteration. All of the cases were related to anoxic events.


The Lennox-Gastaut syndrome: electroencephalographic characteristics, clinical correlates, and follow-up studies.

Electroencephalographic and clinical findings are reported for 100 patients with the Lennox-Gastaut (LGS) triad of slow bilateral spike and wave (BSW), retardation and multiple seizures. Neurological and mental deficits were frequently observed, especially in patients who developed seizures before age 1 yr. More than half of the patients had focal epileptiform discharges that peaked in occurrence at age 4-6 yrs. EEG follow-up showed that background frequency slowed when patients developed the LGS pattern, and increased after recovery. Only 2 patients developed normal EEGs on follow-up, although 22 patients no longer showed the LGS pattern after an average of 3 yrs 3 mos follow-up.


Surface sphenoidal electrode for recording anterior temporal spikes.

Spike analysis was performed to determine if surface sphenoidal electrodes were suitable substitutes for depth sphenoidal or anterior temporal electrodes in outpatient EEG recordings for the diagnosis of complex partial seizures of anterior temporal origin. Spike measurements consisted of spike detection rate, spike amplitude, and location of maximal amplitude spikes. Depth sphenoidal electrodes had the highest yield in these three measurements. Surface sphenoidal electrodes did not differ from anterior temporal electrodes in spike detection rate and spike amplitude, but the former recorded almost no maximal amplitude spikes, while the latter had approximately 30% of the maximal spikes. It is concluded that surface sphenoidal electrodes are slightly inferior to anterior temporal electrodes, but the differences between them are minimal for practical purposes in outpatient EEG recordings.


Frontal spindle activity that appears in conjunction with nontraumatic diffuse encephalopathy.

This peculiar 11-14 Hz spindle activity appears predominantly in the frontal area, and was observed in eight patients with impaired consciousness caused by nontraumatic diffuse encephalopathy. Characteristic of this frontal spindle activity is its transience and accordance with changes in the arousal level of the patient. When the degree of impaired consciousness in the patient was minimal and clinically not very apparent, this spindle activity appeared during light drowsiness. In lethargic patients, it was observed when the patient's level of consciousness rose (e.g. immediately after opening and closing the eyes). These frontal spindles disappeared at the onset of Stage 2 sleep, when normal physiologic spindle waves that are dominant in the vertex area appeared. A paroxysmal discharge was sometimes recorded in association with the frontal spindle activity and it disappeared at about the same time as these spindles. The prognosis was satisfactory for all patients in whom frontal spindle activity was observed; its correlation to spindle coma is also studied.


Neurophysiologic studies in nightmare sufferers.

Nightmares have long attracted neurologic and psychiatric attention, yet little is known of their pathophysiology. We recorded 17-channel electroencephalograms (EEGs), brainstem auditory evoked potentials (BAEPs), long-latency auditory event-related potentials (AEPs), and overnight cassette sleep EEGs (AEEGs) in 10 individuals with recurrent nightmares. They were all nocturnal sleepers, took no medications, do not abuse alcohol or drugs, and had no known medical or psychiatric illnesses. Five patients were being evaluated for other complaints, 3 reported disturbed nocturnal sleep and daytime sleepiness, and 2 sought attention chiefly for nightmares. All 10 patients had normal EEGs and BAEPs. BAEP latencies did not differ significantly from control subjects. Latencies and amplitudes of AEPs were not significantly different in nightmare sufferers and controls, but the former had higher amplitude N100, P160, and N200. Those patients with sleep complaints had on overnight AEEG, less sleep, decreased slow-wave sleep, and more awakenings than those without sleep complaints, but nightmares did not occur during the AEEG recordings.


Risk factors for coronary artery disease in lean and obese women with the polycystic ovary syndrome.

The evidence that some women with the polycystic ovary syndrome (PCOS) are hyperinsulinaemic has brought into question their risk of developing early coronary artery disease. We have focused on three cardiac risk factors which have been associated with hyperinsulinaemia by measuring glucose tolerance, fasting serum lipid concentrations and blood pressure in women with PCOS. Comparison of clinical and biochemical measurements in lean and obese women with PCOS and in women with normal ovaries. Determinants of the risk factors for coronary artery disease were assessed by multiple regression analysis. One hundred and two women with ultrasound diagnosed PCOS and 19 lean women with normal ovaries were studied. Patients were recruited from a reproductive endocrine clinic. Fasting total cholesterol, triglycerides, high density lipoproteins (HDL), HDL2, glucose tolerance, fasting and stimulated insulin, gonadotrophins, testosterone and androstenedione were measured during a 2-hour oral glucose tolerance test. Recumbent blood pressure was measured automatically. Lean women with PCOS were found to be hyperinsulinaemic and have reduced serum HDL and HDL2 concentrations compared to women with normal ovaries; serum insulin concentrations correlated positively with plasma glucose and blood pressure measurements in multiple regression analysis. Obese women with PCOS were in addition found to have higher systolic blood pressure, serum triglyceride and plasma glucose concentration than lean women with PCOS and controls. These results support the evidence that hyperinsulinaemic women with PCOS have an increased risk of developing cardiovascular disease and therefore form a population in whom metabolic screening is advisable.


How common are polycystic ovaries in normal women and what is their significance for the fertility of the population?

We wished to determine the prevalence of polycystic ovaries (PCO) and relate morphological appearance to fertility. We sent postal invitations to a random sample of women born in the years 1952-1969 from a list of a single Group Practice to attend for reproductive history questionnaire, examination, ultrasound scan of the ovaries and hormone measurements within 5 days of the onset of menstruation. Of 1065 women potentially available for study, 571 (54%) replied of whom 353 (62%) agreed to participate. One hundred and ninety (18%) completed the study, 163 were deferred (57 because of current or very recent pregnancy, 106 because of inconvenience at time approached), and 18 additional women volunteered. Prevalence of polycystic ovaries, ovarian size and morphology, menstrual history, features of androgen excess, fertility status, serum hormone levels. The prevalence of PCO was 22% (41/190). PCO and non-PCO women were similar with respect to age, body mass index, oral contraceptive pill (OCP) usage, acne, and menstrual pattern but hirsutism (Ferriman and Gallwey score > 7) was significantly (P = 0.006) more frequent among PCO women. Proven prior fertility was the same in PCO (56%) and non-PCO (64%) women and an equal proportion in each group had not yet tested their fertility. Of those women with previously proven fertility, self-perceived difficulty in conception occurred in similar proportions of women with and without PCO. Unresolved primary or secondary infertility (2.5-4%) was similar in both groups. Ovarian volume (each ovary separately) was larger in women with PCO irrespective of current OCP usage. Serum levels of oestradiol and FSH were similar in PCO and non-PCO women, but LH was distributed around a higher median in PCO women. Median testosterone and androstenedione levels were the same in PCO and non-PCO women. The prevalence of polycystic ovarian morphology is high but, in this sample of women, was accompanied by minimal clinical manifestations and apparently no deleterious effects on earlier fertility. An isolated finding of polycystic ovaries may be a normal variation and should not necessarily imply altered fertility potential.


The IgG subclass distribution of TSH receptor blocking antibodies in primary hypothyroidism.

TSH receptor stimulating antibodies are restricted to the IgG1 subclass suggesting an oligoclonal origin. We wished to determine whether thyroid stimulation blocking antibodies, which also bind to the TSH receptor but may cause hypothyroidism, are similarly IgG subclass restricted. Sera containing TSH receptor blocking antibody activity were separated into IgG subclasses by negative depletion of all other subclasses on affinity columns of subclass-specific monoclonal antibodies or protein A as appropriate. Eleven patients from two centres were studied. All had autoimmune hypothyroidism and TSH receptor blocking antibodies but no thyroid stimulating antibodies. TSH receptor blocking antibody activity was measured by assessing inhibition of TSH-stimulated cAMP production by human thyroid cells (five Israeli samples) or by the FRTL-5 rat thyroid cell line (six Korean samples). IgG1 was the most important subclass containing TSH receptor blocking antibody activity but complete restriction to this subclass was never seen. Clearly detectable activity was found in the IgG2 subclass in eight patients, in the IgG3 subclass in three patients, and in the IgG4 subclass in six patients. The percentage recovery of activity in the sum of the separated fractions generally corresponded to the activity in whole immunoglobulin, being 117 +/- 66% in the nine patients in whom this could be assessed, although in one of these, the activities in the sum of the fractions was much higher (284%). Unlike thyroid stimulating antibodies, thyroid stimulation blocking antibodies are not subclass restricted and are therefore likely to have a polyclonal origin. The IgG subclass distribution of these blocking antibodies resembles that of thyroglobulin and thyroid peroxidase antibodies.


Insulin secretion, insulin sensitivity and hepatic insulin extraction in primary hyperparathyroidism before and after surgery.

Primary hyperparathyroidism (pHPT) is associated with hypertension, hyperinsulinaemia, and insulin resistance. The present study investigated the causes of these metabolic disturbances by quantifying insulin sensitivity and glucose effectiveness, and by assessing the time course of beta-cell insulin secretion and hepatic insulin extraction, during a dynamic condition such as after an intravenous glucose load. In addition, we evaluated the possible link between metabolic disorders and high blood pressure. We studied 16 patients with pHPT, before and 12 weeks after parathyroidectomy; eight of these patients were re-evaluated one year after surgery. The control group consisted of 18 healthy volunteers. All subjects underwent an oral and a frequently sampled intravenous glucose tolerance test. The data from the intravenous glucose tolerance test were analysed by means of the minimal model technique which yields relevant parameters to comprehend the metabolic status of the single individual. The glucose intolerance condition was characterized by a severely impaired insulin sensitivity in pHPT (3.2 +/- 0.5 vs 9.5 +/- 1.5 x 10(4)/min/(microU/ml) of control subjects; P < 0.001), as well as by a reduced glucose effectiveness, (0.02 +/- 0.002 vs 0.03 +/- 0.003/min of control subjects; P < 0.04). Total insulin secretion during the 4 hours of the test was almost twofold elevated in comparison to the control subjects (32795 +/- 4769 vs 16864 +/- 1850 pM, P < 0.004) and its basal component significantly correlated with the high blood pressure. Hepatic extraction of insulin was significantly increased in pHPT (85 +/- 2 vs 76 +/- 2%, P < 0.03), possibly as a compensatory mechanism of hypersecretion, which however did not prevent peripheral hyperinsulinaemia in pHPT. Patients with pHPT were divided into two subgroups with normal and impaired glucose tolerance. The patients with impaired glucose tolerance had a significant reduction of first phase insulin response, although their basal and stimulated insulin levels were higher. Tissue insulin sensitivity and glucose effectiveness did not significantly differ between the two subgroups. After surgery, all the biochemical parameters (former hypercalcaemia, hypophosphataemia, elevated parathormone levels) were normalized, insulin sensitivity significantly improved (6 +/- 1 x 10(4)/min/(microU/ml), P < 0.001), whereas glucose effectiveness remained completely unchanged. Basal and stimulated insulin responses were insignificantly lowered after surgery, and hepatic extraction did not change either. Patients with pHPT exhibited decreased insulin sensitivity and insulin hypersecretion. The latter is only partially ameliorated by increased hepatic insulin extraction. After surgery, although the biochemical abnormalities were fully reversible, the metabolic changes improved only partially.


Effectiveness of computer-assisted perimetry in the follow-up of patients with pituitary microadenoma responsive to medical treatment.

Patients were studied before and after 1 year of bromocriptine or 6 months of SMS 201-995 treatment, for prolactinomas or GH-secreting adenomas, respectively. Seventeen patients with intrasellar pituitary tumour (ten prolactinomas, all females; seven GH-secreting adenomas, four males and three females) and the presence of relative or absolute scotomas, were examined. We used computed tomodensitometry, Goldman perimeter and computer-assisted perimetry. The patients were divided into three groups according to their response to medical treatment as proved by computed tomodensitometry which revealed the disappearance of the tumour in four prolactinomas (group 1), a reduction > 40% in three prolactinomas and in three acromegalics (group 2) and no significant variation in the diameter of the adenoma in three prolactinomas and in four acromegalics (group 3). Comparison by the paired t-test of the visual fields before and after treatment revealed a significant positive change (P < 0.01) for all patients in groups 1 and 2 and for one patient in group 3, with disappearance of the scotomas in all cases in group 1 and in two cases in group 2. Visual field defects were detected by means of the Goldman perimeter in only one patient with prolactinoma and in two acromegalics, although the computer-assisted perimetry showed that, in 15 out of 17 patients, visual impairment was unilateral and in all cases the presence of relative scotomas was concentrated in the upper temporal quadrant. The visual defects observed with computer-assisted perimetry and the pituitary tumour dimension evaluated with computed tomodensitometry did not show significative correlations (r = 0.059, P NS). Computer-assisted perimetry was most useful in the diagnosis and follow-up of patients with pituitary adenoma, especially in the evaluation of small masses without subjective symptoms of visual loss, when the Goldman perimeter does not usually allow us to recognize minimal chiasmatic involvements or the improvement of visual field as a result of the medical therapy.


Analysis of trough serum growth hormone concentrations: comparison of an immunoradiometric assay and a sensitive ELISA for growth hormone.

We compared a sensitive assay for GH (ELISA) with a conventional immunoradiometric (IRMA) assay with particular reference to the oscillatory activity detected by Fourier transformation and the estimation of trough concentrations using occupancy analysis. Eight healthy adult male volunteers underwent 24-hour profiles during which samples were drawn at 20-minute intervals. Samples were analysed by an ELISA and an IRMA system. The 24-hour serum GH concentration profiles were subjected to Fourier transformation and to occupancy analysis. No additional GH periodicities could be determined in the ELISA data other than the well documented 180-200-minute periodicity. Median observed concentrations (OC) at 5% occupancy were 0.035 mU/l (range 0.004-0.22) for the ELISA and 0.035 mU/l (range 0.001-0.50) for the IRMA. For all OC parameters, 5, 50 and 95%, there was a good correlation between the ELISA and IRMA systems. The mean difference (bias) between the ELISA and IRMA were -0.05, -0.28 and -1.40 mU/l at OC values of 5, 50 and 95% respectively and the standard deviations of the difference at the same OC values were 0.10, 0.50 and 1.61 mU/l. Although there is a qualitative improvement on visual inspection of individual 24-hour serum GH profiles obtained using the ELISA system, there is little additional information gained in terms of pulse periodicity or occupancy analysis.


Serum levels of growth hormone-binding protein and insulin-like growth factor-I during puberty.

The aim was to investigate the effect of pubertal development on serum levels of growth hormone binding protein (GHBP) and IGF-I, and to study the relationship between GHBP levels and height standard deviation score (SDS), nutritional state and IGF-I levels. The investigation was performed on serum samples from 72 healthy adolescents of different pubertal stage. Results were compared to those obtained in 46 prepubertal children. Serum levels of GHBP were measured by HPLC gel filtration and IGF-I levels were measured by RIA after acid-ethanol extraction. No effect of pubertal stage on serum levels of GHBP was found. A positive relationship was found between serum levels of GHBP and height SDS (r = 0.38; P < 0.005) and weight expressed as percentage of median weight for height age (r = 0.46; P < 0.0005). Serum levels of IGF-I increased during puberty and were not correlated with height SDS or weight for height age. In pubertal subjects, no relationship existed between serum levels of GHBP and IGF-I. In prepubertal subjects, however, a significantly positive relationship between GHBP and IGF-I levels (r = 0.66; P < 0.0005) was found. Pubertal development does not seem to influence serum levels of GHBP. Height SDS and nutritional state are related to the concentration of GHBP. Before puberty, the level of GHBP is positively related to IGF-I levels; during puberty, however, the increase in serum IGF-I levels is not accompanied by changes in the amount of circulating GHBP.


The sensitivity of growth hormone secretion to medical treatment in acromegalic patients: influence of age and sex.

We investigated the relationship between age, sex, pituitary tumour volume, serum GH, PRL and IGF-I levels with the responsiveness of GH to TRH, bromocriptine and octreotide in patients with acromegaly. We performed a retrospective study. Correlations were determined between all variables using univariate regression analysis. One hundred previously untreated acromegalic patients were studied (60 males (age 23-76; mean 49 years) and 40 females (age 25-83; mean 51 years)). We studied tumour volume, fasting morning circulating levels of GH, PRL and IGF-I, mean 24-hour circulating GH levels and the acute GH responses to TRH, bromocriptine and octreotide. Tumour size was related to serum and mean 24-hour GH levels, but not to IGF-I. Circulating IGF-I and GH levels were related only for the group of patients whose fasting and unsuppressed GH level was 80 mU/l (40 micrograms/l) or less. Older patients tended to have lower circulating GH and IGF-I levels. There was a close similarity in the responsiveness of tumorous GH secretion to TRH, bromocriptine and octreotide. An elevated serum PRL level predicted a stronger inhibitory response of bromocriptine on GH. The sensitivity of GH release to octreotide was highest in elderly (especially male) acromegalics, as well as in patients with lower IGF-I levels. Hormone secretion by GH secreting pituitary tumours, as well as circulating IGF-I levels, tend to be lower in elderly patients. These tumours are more sensitive to octreotide, especially in elderly male patients. This suggests that octreotide might be used especially successfully as a primary medical therapy in elderly, male acromegalics.


Growth hormone secreting pituitary carcinoma: a case report and literature review.

Only five cases of growth hormone secreting pituitary carcinoma have been documented. We present a 49-year-old West Indian male with grossly elevated plasma growth hormone (760-10,400 mU/l), and a large aggressive pituitary tumour that continued to grow despite repeated pituitary surgery, radiotherapy and medical therapy (bromocriptine and somatostatin analogue). Thirteen years after diagnosis the patient died secondary to left ventricular failure. A post-mortem revealed a large locally invasive pituitary tumour, but in addition numerous tumour seedlings within the cerebrospinal fluid space, and a solitary intraparenchymal tumour deposit within the right temporal lobe, clearly separate from the primary tumour. Pituitary carcinoma should be considered in any acromegalic with grossly elevated plasma growth hormone levels who fails to respond to conventional therapy.


Endogenous digitalis-like factors.

The postulate of a natriuretic factor inhibiting the sodium pump in the kidney led to the detection of increased concentrations of endogenous digitalis-like factors in blood after salt loading, in essential hypertension, in pregnancy-induced hypertension and in chronic hypervolaemia. The recent isolation of ouabain or a close isomer thereof from human plasma and the demonstration of a compound similar if not identical to digoxin in adrenals and human urine shows that mammals like non-vertebrates and toads may synthesize cardiac glycosides in their adrenals and possibly in hypothalamus. The hypothalamus also forms other compounds of unknown structure which bind to the cardiac glycoside receptor site. The differential functions of endogenously formed ouabain and of a digoxin-like substance are unclear. The detailed knowledge of the physiological role of both endogenously formed cardiac glycosides in the regulation of blood pressure has still to be worked out.


Factors associated with acute salt-sensitivity in borderline hypertensive patients.

The acute sensitivity to sodium loading has been investigated in 26 borderline hypertensive patients (BHT) undergoing acute i.v. NaCl infusion. Measurements included blood pressure (BP), forearm vascular resistance (FVR) and venous distensibility (VV30), plasma renin activity (PRA), plasma aldosterone, plasma atrial natriuretic factor (ANF), and plasma levels of endogenous Na+/K+ATPase inhibitor. Sodium loading was associated with a greater than 8% increase in mean BP in 12 patients defined as salt-sensitive (NaCl-SENS) in comparison to salt-insensitive (NaCl-INSENS) subset. NaCl-SENS patients in comparison to NaCl-INSENS exhibited 1) a greater baseline VV30 (2.1 vs 1.4 ml/100 ml; p less than .005), and a response to saline characterized by 2) increased FVR (21.4 vs -6.5%; p less than .005), 3) blunted PRA suppression (-42 vs -67%; p less than .05), 4) delayed ANF response and 5) release of a Na+/K+ATPase inhibitor. Post-loading cumulative urinary sodium excretion was reduced in NaCl-SENS borderline hypertensives compared to NaCl-INSENS (2.6 vs 3.8 mumol/min/Kg; p less than .05). We conclude that acute salt-sensitivity in BHT is characterized by a blunted hormonal response to sodium loading which could be responsible of the activation of hemodynamic as well as humoral mechanisms leading to progressive blood pressure increase.


An indirect evaluation of the effect of the autonomic nervous system following converting enzyme inhibition in hypertension.

Common carotid blood flow and cold pressor test were evaluated in 16 patients with sustained essential hypertension before and after 30 days treatment with the converting enzyme inhibitor Enalapril (20 mg). Enalapril decreased blood pressure and carotid vascular resistance with no significant change in heart rate. After treatment, despite a wide range of the responses, the changes in systolic blood pressure to cold test were significantly attenuated, whereas the heart rate responses were not. Acute random and double blind administration of either Cadralazine or Nitrendipine, two vasodilating drugs which are known to cause an activation of the autonomic nervous system, were performed before and after long term treatment by Enalapril. Whereas the blood pressure and heart rate responses to cold test was unmodified by these compounds before Enalapril treatment, significant changes were observed after converting enzyme inhibition: Cadralazine reduced the heart rate response whereas Nitrendipine increased it significantly. The study provides evidence that converting enzyme inhibition causes sympatho-inhibitory influences which are principally observed in stress conditions, with heterogeneous responses depending on the nature and the type of stimulation.


Protein kinase C and cell proliferation in spontaneously hypertensive rats.

Cultured aortic fibroblasts from spontaneously hypertensive rats (SHR) exhibit increased proliferation rate compared with cells from normotensive Wistar Kyoto (WKY) rats. The present study was designed to investigate whether this growth abnormality could be accounted for by alteration in protein kinase C (PKC). The enzyme activation by 12-O-tetradecanoyl phorbol 13-acetate (TPA) promoted 3H-thymidine incorporation which was higher in SHR-derived fibroblasts compared with WKY-derived cells. Likewise, 3H-phorbol 12,13-dibutyrate (PDBu) binding to intact cells was markedly increased in SHR-derived fibroblasts. These findings suggest a difference in PKC activity between the two cell types. In both cell types, serum-induced 3H-thymidine incorporation was enhanced by PKC down-regulation, which was obtained by prolonged treatment of cells with high dose of TPA, whereas it was inhibited in a dose-dependent manner by activation of the enzyme. The changes in serum-induced 3H-thymidine incorporation elicited either by activation or desensitization of PKC, did not differ between SHR and WKY fibroblasts. Our results indicate therefore i) that in the presence of serum PKC exerts an antiproliferative effect in rat aortic fibroblasts and ii) that the increase in PKC activity and in sensitivity to TPA exhibited by SHR-derived fibroblasts, is not involved in the increased proliferation rate displayed by SHR-derived fibroblasts in serum-containing medium.


Hormonal aspects of the relation of liver cirrhosis to essential hypertension.

The association of liver cirrhosis with arterial essential hypertension has been previously described. The present study extends the previous reports by investigating the hormonal relationships that may occur in patients with established essential hypertension associated to liver cirrhosis. We studied the renin-angiotensin, the adrenergic systems and other vasoactive hormones such as arginine-vasopressin, atrial natriuretic peptide, endothelin and parathyroid hormone in cirrhotic patients with and without essential hypertension. The data suggested that the coincidence of arterial hypertension in cirrhotic patients was characterized by the following findings: a decreased renin-angiotensin activity; a reduced systemic vasodilatation; an increased peripheral pressor effect of vasoactive hormones and an increased effective blood volume.


Haemodynamic and metabolic effects of short term administration of synthetic sex steroids in humans.

Synthetic sex steroid administration is a major cause of iatrogenic hypertension but little is known of the haemodynamic or metabolic consequences of these steroids. This study examined the short term blood pressure, volume and metabolic consequences of 5 day administration of synthetic androgen to normal men and synthetic oestrogen or progestogen to normal women. Healthy subjects (8 women, 6 men) on a constant diet took part in each of 3 studies. Males received testosterone undecanoate 120 mg/day (n = 6) and females either ethinyloestradiol 0.3 mg/day (n = 5) or norethisterone 15 mg/day (n = 6) for 5 days in the last week of the cycle. Norethisterone increased lying (+7 mmHg) and standing (+8 mmHg) systolic pressure but the other steroids did not alter blood pressure. All 3 treatments increased body weight. There were no consistent changes in plasma electrolytes or glucose with any steroid, and no urinary sodium retention or changes in urine Na:K ratio. Haematocrit fell on ethinyloestradiol but no steroid significantly increased plasma volume (measured as volume of distribution of 125I human serum albumin). Renin substrate and cortisol rose and renin concentration fell on ethinyloestradiol. These studies suggest that the progestogen component may contribute to the blood pressure raising effects of oral contraceptives.


Renal renin secretion rate and norepinephrine secretion rate in response to centrally administered angiotensin-II: role of the medial basal forebrain.

The influence that centrally administered angiotensin-II (ANG-II) and saralasin (SAR) has on renal norepinephrine secretion rate (NESR) and renal renin secretion rate (RSR) were studied. Rats were given thermal lesions of the medial basal forebrain (MBF) or sham surgery. Twenty-four hours later the right kidney was vascularly isolated (but neurally intact) and perfused with an artificial plasma at either a constant pressure (100 mm Hg) or constant flow (600 microliters/min). Renal perfusate was collected before (pre-injection) and at 10 min intervals after central administration of peptides for determination of NESR and RSR. In both perfusion models, intracerebroventricular (ICV) ANG-II increased renal NESR. In MBF lesioned rats pre-injection renal NESR is reduced and the response to ICV ANG-II is blocked. In both perfusion models ICV ANG-II decreases renal RSR. Concomitant administration of SAR blocks the effect of ANG-II on both NESR and RSR. MBF lesioned rats had significantly elevated pre-injection levels of RSR and there is no change in RSR following ICV ANG-II. These experiments indicate that centrally administered ANG-II increases renal NESR concomitant with a decrease in renal RSR and that MBF lesions block those changes.


Putative monosomy 21 in two patients: clinical findings and investigation using fluorescence in situ hybridization.

Complete monosomy 21 is claimed to be a rare chromosomal disorder in which the cytogenetic investigation is bedevilled by technical difficulties. We describe the disparate clinical features in two patients in whom an initial diagnosis of monosomy 21 was made by routine karyotyping. Fluorescence in situ hybridisation (FISH) confirmed a translocation of chromosome 21 material to the short arm of chromosome 5 and to the X chromosome, respectively. The usefulness of FISH in the investigation of subtle chromosomal rearrangements is hereby demonstrated. These findings also cast doubt on the existence of "pure" monosomy 21 as an entity, and suggest that partial monosomy 21 is a more likely occurrence.


Congenital hypoparathyroidism, seizure, extreme growth failure with developmental delay and dysmorphic features--another case of this new syndrome.

A 4-year-old Saudi female child with extreme failure to thrive, striking dysmorphic features, developmental delay, congenital hypoparathyroidism, UTI, seizures, chronic otitis media, chronic non-specific gastroenteritis and repeated life-threatening infections was followed from birth. She was the product of first-cousin consanguineous marriage. She had striking facies with frontal prominence, deep-set eyes, depressed nasal bridge, beaked nose, long philtrum with thin upper lip, micrognathia, large floppy ears, bifid uvula, and growth retardation with SD score less than -2 for height, weight and head circumference. We believe these features which include congenital hypoparathyroidism, severe growth failure and developmental delay in the absence of chromosomal abnormality represent a newly described genetically determined syndrome.


X chromosome inactivation patterns in haematopoietic cells of female carriers of X-linked severe combined immunodeficiency determined by methylation analysis at the hypervariable DXS255 locus.

The patterns of X chromosome inactivation were determined in 14 females from three unrelated X-linked severe combined immunodeficiency (XSCID) pedigrees. All the females were found to be heterozygous for the hypervariable DXS255 locus, enabling analysis of differential methylation of this locus in peripheral blood haematopoietic cells. All six obligate carriers manifested a unilateral X chromosome inactivation in the T lymphocyte population. Differential methylation analysis of T lymphocytes was subsequently applied to establish the carrier status of females at risk in the XSCID pedigrees. In the B lymphocyte population of four XSCID carriers a unilateral X chromosome inactivation was observed. Four other carriers had minor fractions and one carrier had a substantial fraction of B lymphocytes with the XSCID gene defect on the active X chromosome. Within single XSCID pedigrees the carriers manifested different patterns. In two pedigrees the granulocyte populations of all carriers showed a random distribution of X chromosome inactivation. In the third pedigree the granulocytes of the three carriers analyzed manifested complete inactivation of the X chromosome that carried the XSCID mutation, exposing a selective disadvantage of granulocytes that express the XSCID defect. The pedigree-dependent differences in the involvement of the granulocyte population suggest the existence of two distinct XSCID defects.


Prevalence of retinitis pigmentosa in Slovenia.

Two hundred and twenty-nine symptomatic patients with retinitis pigmentosa were ascertained in Slovenia between 1986 and 1990. Twenty-three further patients were identified while data from 63 families (82 patients) were being collected. After correction for underascertainment, a prevalence of 1 in 6023 was estimated in the Slovene population (1,999,477 in 1990). The highest prevalence of 1 in 1902 was found in the age group 65 years and older. Of 63 analysed families, 17 (27%) showed autosomal dominant, 13 (21%) autosomal recessive, and one family (1.5%) X-linked inheritance; in 30 families (47.5%) isolated cases were found; and in two families the mode of inheritance was impossible to determine.


Reassessment of a chromosome 12q+ marker by fluorescent in situ hybridization (FISH).

We present a case previously described by Jenkins et al. (1983) as atypical Down syndrome (DS). The initial diagnosis was first made on the basis of phenotypic and cytogenetic data. This analysis was supported by studies of superoxide dismutase (SOD1) activity that maps to band 21q22.1. Results from phenotypic, chromosome banding and SOD1 studies suggested a karyotype of 46,XX,-12,+t(12pter to 12qter::21q21 to 21q22.?2). Using fluorescent in situ hybridization (FISH) for chromosome painting with DNA libraries derived from sorted human chromosomes to stain selectively the chromosomes No. 21 and No. 12, we demonstrate that the marker chromosome 12q+ has no chromosome 21 content but it is derived from chromosome 12.


Leber hereditary optic neuropathy: estimation of number of embryonic precursor cells and disease threshold in heterozygous affected females at the X-linked locus.

LHON has been suggested to involve both mitochondrial and X-chromosome-linked loci. By extending two-locus mitochondrial and nuclear gene analytic methods, we recently proposed that a proportion of affected females are likely heterozygous at the X-linked locus and affected due to unfortunate X-chromosome inactivation. Assuming that the optic tissue is the primary site of action of the mutant gene(s), we further propose here that there should be no fewer than six embryonic precursor cells for the involved optic tissue at the stage in early development when X-chromosome inactivation occurs. We also estimate that the disease threshold (i.e. proportion of cells with abnormal X-chromosome active in the responsible tissue at the time of X-chromosome inactivation) for a heterozygous female is in the range of 0.60 to 0.83.


Reproductive behaviour following spontaneous loss of pregnancy after prenatal diagnosis.

One hundred and fifty-eight women of advanced maternal age with complete follow up who experienced spontaneous fetal loss after prenatal diagnosis were studied for reproductive behaviour as well as prenatal diagnosis in a subsequent pregnancy. A higher rate of subsequent pregnancies amongst women who experienced an early spontaneous abortion after chorionic villus sampling (CVS) was expected compared with women who lost a pregnancy later during pregnancy after amniocentesis. Of the 92 women who underwent CVS in a previous pregnancy, 57 (62%) became pregnant again. Of the 66 women who underwent amniocentesis in the pregnancy that ended in fetal loss, 34 women (52%) had a subsequent pregnancy. The cumulative incidence of subsequent pregnancies was significantly influenced by maternal age but not by parity or the method of prenatal testing. Most women who decided on a new pregnancy opted for prenatal diagnosis. There was a preference for amniocentesis if the patient had previously undergone CVS. However, the reverse was not the case.


Autosomal recessive microcephaly with early onset seizures and spasticity.

We describe two siblings, a male and a female pair, born of consanguineous parents, affected with a rare genetic form of congenital microcephaly. The clinical syndrome is characterized by early onset myoclonic seizures, spasticity, and profound psychomotor retardation without detectable brain malformations. To date, only two kindreds and one sporadic case with a similar clinical picture have been observed and reported (Tolmie et al. 1987, Bundey & Griffiths 1977). The severity of the neurological features and their perinatal onset differentiate the syndrome from the more common autosomal recessive microcephaly with spasticity/seizures.


The gene for Best's macular dystrophy is located at 11q13 in a Swedish family.

A large Swedish family with more than 250 cases of Best's macular dystrophy has been clinically and genetically studied. The gene was traced to a couple born in central Sweden in the 17th century. Highly significant evidence for genetic linkage to DNA markers on chromosome 11q13 was detected. A lod score of 15.12 was obtained at recombination fraction 0.01 with DNA marker INT2 (also called FGF3). The retinally expressed gene ROM1, which maps to the same chromosomal region is a candidate for this genetic disease.


Phospholipid-containing toxic malaria antigens induce hypoglycaemia.

Hypoglycaemia is associated with severe malaria and is an important prognostic indicator. Molecules liberated during overnight incubation of erythrocytes infected with Plasmodium yoelii induce marked hypoglycaemia in normal mice, with a delayed time course compared with insulin; some, though weaker, activity could also be obtained by overnight incubation of uninfected erythrocytes. The active component shares many properties with the phospholipid-containing molecules which we have previously shown to be toxic and to induce the release of tumour necrosis factor (TNF) from macrophages. However a MoAb which neutralizes the cytotoxicity of tumour necrosis factor in vitro did not prevent this induction of hypoglycaemia, whereas antiserum against the toxic antigens did, as did immunization of normal (but not the immunoglobulin-deficient SCID) mice with the same material. Furthermore, normal mice injected with the antigens after immunization with phosphatidyl inositol or inositol monophosphate did not develop hypoglycaemia; the latter compound was also inhibitory when mixed with the antigens before injection. These compounds were previously shown to block the induction of TNF by the antigens and to induce the production of inhibitory antibodies. The role of these molecules in the etiology of the hypoglycaemia of malaria is discussed.


Plasma concentration of IL-6 in systemic lupus erythematosus; an indicator of disease activity?

To investigate the possible role of IL-6 in the activation of the autoimmune process in systemic lupus erythematosus (SLE), we serially measured concentrations of IL-6, IgG, and anti-dsDNA antibodies before and during exacerbations in patients with SLE. In addition, we serially related the IL-6 response to the generation of the acute phase reactant C-reactive protein (CRP). Sixteen consecutive patients who developed an exacerbation were analysed in this study. Blood samples were drawn in EDTA monthly. At the time of maximal disease activity during exacerbation, IL-6 plasma concentrations were increased (greater than or equal to 6 pg/ml) in 12 out of the 16 cases. Concentrations of IL-6 correlated with the concentrations of CRP (P less than 0.01) and the score of the disease activity index (P less than 0.05). No correlation was found between IL-6 concentrations and concentrations of anti-dsDNA or IgG. The course of changes in IL-6 concentrations before the exacerbation was variable. Five out of the 16 exacerbations studied were characterized by a prominent rise of IL-6 at the time of maximum disease activity. In this subgroup serositis as well as elevated concentrations of CRP were observed more frequently (P less than 0.02). Seven exacerbations were not accompanied or preceded by changes in IL-6 concentrations and showed generally low IL-6 concentrations. In this latter subgroup cerebral involvement was seen more frequently (P less than 0.02). Our data do not suggest a pathogenic role for IL-6 in the generation of IgG and/or anti-dsDNA antibodies before exacerbations. Rises of IL-6 concentrations before exacerbations in SLE seem only to occur in a subgroup of patients with SLE characterized by the presence of serositis and elevated concentrations of CRP during exacerbation.


Hodgkin's cells express a novel pattern of adhesion molecules.

Adhesion molecules play an important role in the functioning of the immune system, particularly with regard to cell-cell interactions and antigen presentation. Several adhesion molecules are expressed on Hodgkin's disease-derived cell lines and these are important in their molecular interactions as antigen presenting cells (APC). There are no data regarding the expression of many of these adhesion molecules on Reed-Sternberg cells and its mononuclear variant (Hodgkin's cells (HC)) present in pathological material. To obtain this information we undertook an immunohistological study on material from 18 cases of Hodgkin's disease using a panel of MoAbs to examine the expression of adhesion molecules on HC. The HC were shown to express the integrin beta 1 subfamily molecules, LFA-1 (CD11a) and p150,95 (CD11c) in high density but lacked CR3 (CD11b). All of the immunoglobulin gene superfamily adhesion molecules studied were present to some degree on HC, with ICAM-2, in particular, showing moderate to strong expression in most cases. The Hermes antigen CD44 was present in high density but leukosialin (CD43), another molecule present on diverse leucocyte types, was, in general, not detected on HC. These new data showing that ICAM-1, ICAM-2 and LFA-3 are, like LFA-1, expressed on HC emphasize the ability of HC to act as APC. The known adhesion molecule phenotype of the recently defined haematopoietic lineage of human dendritic cells (DC) is broadly similar to that of HC, perhaps supporting the hypothesis that some HC represent a malignancy of an APC (DC) lineage.


Control of immune-mediated disease of the central nervous system requires the use of a neuroactive agent: elucidation by the action of mitoxantrone.

Mitoxantrone was used as an immunosuppressive probe to elucidate a means for the control of experimental allergic encephalomyelitis (EAE) induced in Biozzi AB/H mice following injection of spinal cord homogenate emulsified in Freund's adjuvant. A single i.p. injection of 2.5 mg/kg of mitoxantrone, 1-2 days before the anticipated onset of EAE, failed to prevent the majority of animals from developing clinical disease, whereas when the compound was injected directly into the central nervous system (CNS), at this time point, significantly increased therapeutic benefit was evident, with most animals failing to develop clinical EAE. Although the clinical use of intrathecal mitoxantrone is strongly contraindicated, these data suggest that increased therapeutic benefit may be achieved in immune-mediated disease of the CNS by targeting immunosuppressive doses of suitable agents, on lymphocyte activation within the CNS. In addition, direct administration of immunosuppressive doses into the CNS may reduce potentially unwanted (side) effects in the periphery.


Mesangial sclerotic change with persistent proteinuria in rats after two consecutive injections of monoclonal antibody 1-22-3.

Irreversible mesangial changes with persistent proteinuria were induced in rats given two consecutive injections 2 weeks apart of a MoAb 1-22-3 to rat mesangial cell. The characteristics of the resulting lesions were investigated and compared with those of the reversible change induced by a single injection. At 24 h after the second injection, mesangiolytic changes similar to those after a single injection were evident. The accumulation of macrophage-like cells in glomeruli observed at 1 week after the first injection was not evident during the experimental period after the second injection. Hypercellularity with the characteristics of intrinsic mesangial cell and increased mesangial matrix were already present 1 week after the second injection. And mesangial sclerotic change progressed up to 6 months. Deposition of collagen type I and type III and accumulation of collagen fibril at the ultrastructural level were evident in rats 6 months after the second injection. Proteinuria started immediately and continued for more than 6 months after the second injection. The mesangial sclerotic change with persistent proteinuria described here is considered to be a better model for investigating the mechanism of chronic progression of human mesangial proliferative glomerulonephritis.


Human immunoglobulin production in immunodeficient mice: enhancement by immunosuppression of host and in vitro activation of human mononuclear cells.

The affect of host and donor related factors on successful engraftment of human cells into mice was examined to minimize the variability that has been observed in successful development of human-mouse chimera for the study of human disease and immune physiology and regulation. Human immunoglobulin production in severe combined immunodeficiency (SCID) mice engrafted with human peripheral blood mononuclear cells (PBMC) was augmented by immunosuppressing recipient mice and activating donor PBMC. Immunosuppression of recipient mice with 3 Gy of gamma-irradiation induced a 10-fold increase in human IgG in the sera of engrafted SCID mice. Variation in production of human IgG in recipient mice correlated with preinjection phenotype and activation status of injected PBMC. Mice injected with PBMC with a low CD4/CD8 ratio (less than 0.5) produced no detectable circulating human immunoglobulin. When the CD4/CD8 ratio was greater than 1.5, human IgG was detected in sera of PBMC-recipient SCID mice. Serum IgG increased 10-fold following in vitro activation of donor PBMC with anti-CD3, IL-2 and Staphylococcus aureus. Successful engraftment and serum IgG production was evidenced by an increase in the recovery of activated human IgG+ cells in the spleens of mice with maximal IgG production. Optimization of functional engraftment required modification of both the host (SCID mice) and the donor cells.


Structure and expression of the mb-1 transcript in human lymphoid cells.

The mb-1 gene encodes a protein associated with membrane-bound immunoglobulins (mIg) and it has been suggested that it may play a role in signal transduction. Using a murine probe, we cloned a human complementary DNA homologous to the murine mb-1 sequence. Its complete sequence is in full agreement with a recently published human cDNA sequence but differs from a previously reported partial sequence. We studied mb-1 expression in human cells at various maturation stages. Large amounts of a single 1.4 kb transcript were detectable in B cell lines carrying mIg of the mu, gamma, alpha 1 or alpha 2 isotype. The mb-1 mRNA was also expressed in pre-B cells and in cells expressing truncated membrane mu chains devoid of associated light chains. Only trace amounts of mb-1 mRNA were found in tissue samples containing numerous plasma cells, while no mRNA was detected in several plasmacytoma cell lines, indicating that expression is shut down in plasma cells. No signal was found in T cells or in non-lymphoid tissues. Altogether, these results show that the human mb-1 gene is expressed in pre-B cells and B lymphocytes, regardless of the isotype of the mIg heavy chain and is no longer expressed in plasma cells.


Endoglin: a 180-kD endothelial cell and macrophage restricted differentiation molecule.

A MoAb RMAC8 was generated by immunizing Balb/c mice with cultured human umbilical vein endothelial cells (HUVE). The molecule recognized had a mol. wt of 180 kD non-reduced, 95 kD after reduction and 66 kD in its reduced and N-deglycosylated form. Sequential immunoprecipitation studies with the MoAb 44G4, which recognizes the O- and N-glycosylated homodimer endoglin, showed that both MoAbs recognize the same molecule on HUVE and phorbol myristate acetate (PMA)-stimulated U937 cells. The distribution of the RMAC8-recognized molecule was the same as that described for endoglin, i.e. arterial and venous endothelium, myelomonocytic and pre-B leukaemia cells and cell lines; however, unlike 44G4, RMAC8 also reacted weakly with monocytes and strongly with in vitro differentiated macrophages as well as peritoneal and alveolar macrophages. This distribution of endoglin was confirmed by Northern blot analysis using a full length endoglin cDNA probe. These studies suggest that endoglin is a differentiation marker on macrophages.


Immunophenotypical alterations in a subset of patients with common variable immunodeficiency (CVID).

We investigated the expression of surface molecules on lymphocytes from 20 patients with CVID and 40 healthy subjects. Lymphocytes were analysed by dual colour flow cytometry. We identified a subset of patients (8 of 20) characterized by low CD4/CD8 ratio (less than 1.1), expansion of T cells co-expressing the activation marker HLA-DR and significant increase in CD8+ T cells co-expressing CD57. Expression of the adhesion molecules LFA-3 (CD58) and ICAM-1 (CD54) was significantly increased in this subgroup. In addition, within the CD4+ T cells the percentage of CD29+ (memory) cells was increased, while the CD45RA and LAM-1 (Leu-8) antigens were depressed. These results indicate that in a subgroup of CVID patients T cells are activated in vivo and the CD57+CD8+ lymphocyte subpopulation, supposed to comprise functional suppressor T cells, is expanded. We suggest a chronic viral infection in these patients, but it is not clear whether this is primary or secondary to the underlying defect.


The concentration of the C-type lectin, mannan-binding protein, in human plasma increases during an acute phase response.

Two ELISAs for estimating mannan-binding protein (MBP) were constructed and the concentration of MBP in plasma was followed in patients undergoing major surgery and in patients having a malarial attack. In both cases increases of MBP in the plasma were observed. The relative increase and the kinetics varied from person to person. The concentration of MBP increased between 1.5- and three-fold following surgery. In some patients an increase was seen at day 1 whereas in others the increase was not observed until days 3-9. In the malaria patients an increased level of MBP was maintained during 30 days of treatment with chloroquine. The relative increase in MBP was independent of the presurgery or premalaria levels.


Evaluation of serum levels of tumour necrosis factor-alpha (TNF-alpha) and soluble IL-2 receptor (sIL-2R) and CD4, CD8 and natural killer (NK) populations during infrared pulsed laser device (IPLD) treatment.

The purpose of this study was to evaluate serum levels of TNF-alpha, sIL-2R and distribution of peripheral leucocyte subsets in patients with advanced neoplastic disease undergoing IPLD treatment. Fifteen cancer patients with evidence of persistent disease were further divided in two groups according to outcome at the end of the period of clinical evaluation: group 1 patients were still alive and group 2 patients had died. Our results show: (i) an increase in the initial level of TNF-alpha in both groups; (ii) a decrease in TNF-alpha levels during the follow up of group 1 patients; (iii) a significant increase in serum levels of sIL-2R in patients in group 2 compared with those in group 1; (iv) a progressive and constant increase in TNF-alpha levels in group 2; (v) a decrease in CD4+CD45RA+ subpopulation in both groups; (vi) an increase in CD25+ cells; (vii) an increase in CD4+, CD4+CD45RA+ and CD25+ cells during the follow up of group 2 patients. The data generated here form the basis for further investigations on the use of IPLD as a single agent and in combination with other biological response modifiers in cancer patients.


Alterations in mononuclear cell tumour necrosis factor-alpha (TNF-alpha) response in patients on long term cuprophane haemodialysis.

We have investigated TNF-alpha secretory response of peripheral blood mononuclear cells (PBMC) from 13 uraemic patients undergoing regular haemodialysis with cuprophane membrane (CM). Sixteen healthy subjects and five uraemic patients under conservative therapy were also studied as controls. Cells of haemodialysis patients exhibited increased TNF-alpha release in vitro in the absence of activating stimuli other than culture conditions, as compared with normal and uraemic controls. In contrast to normal cells, this spontaneous secretion of TNF-alpha from dialysis PBMC could not be significantly reduced by addition of polymyxin B to culture medium, thus indicating its independence of trace amount of lipopolysaccharide (LPS) present in the medium as contaminant. Furthermore, predialysis PBMC were considerably more sensitive to stimulation with 10(7) pg/ml of LPS under in vitro culture conditions than normal and uraemic controls. To elucidate a role of direct contact with CM in stimulation of TNF-alpha release from monocytes, PBMC were cultured on CM in vitro. Contact with CM stimulated TNF-alpha secretion from PBMC above the level of cells cultured on tissue culture plastic. This response persisted with time in culture in contrast to a transient LPS-induced TNF-alpha release. Furthermore, PBMC stimulated by contact with CM for 2 days did not lose the capacity to secrete TNF-alpha in response to a subsequent LPS stimulation, while a 2-day treatment of cells with LPS was followed by LPS refractory state. Therefore, direct contact with CM induces in PBMC a long-lasting TNF-alpha response which is not down-regulated by the acquisition of refractoriness in a manner similar to that which occurs in the case of LPS stimulation. These in vitro findings provide a possible explanation of the observation that predialysis PBMC exhibit elevated TNF-alpha secretory capacity.


Haemodialysis as a model for studying endogenous plasma DNA: oligonucleosome-like structure and clearance.

The rate of clearance of extracellular plasma DNA in man has important implications for pathogenetic mechanisms in systemic lupus erythematosus (SLE), as well as for certain other clinical states. Present knowledge of this parameter is derived exclusively from studies of injected, naked DNA in animals. Recent information indicates that the physiologic form of plasma DNA in SLE is that of oligonucleosome-like molecules rather than of naked DNA and consists of multimeric complexes of DNA bound to histone, probably arising from an apoptotic process. In order to study the rate at which these oligonucleosome-like complexes are removed from plasma and to do so in man rather than experimental animals, we exploited the observation that during haemodialysis large amounts of DNA are released, apparently within the dialysis coil, into the patient's plasma. Since this release appears to cease promptly with termination of the procedure, it offered the potential for estimating the rate of removal of such DNA from human plasma. Moreover, if that DNA, as postulated, were shown to possess an oligonucleosome-like structure resembling that found endogenously in human SLE, the relevance of such information to the human disease state would be further enhanced. The present results support the conclusion that DNA released into plasma during haemodialysis possesses such an oligonucleosome-like structure. The plasma half-life of that DNA in man was found not to exceed 4 min. The highly dynamic state thus implied for extracellular endogenous plasma DNA in man has important implications for pathogenetic mechanisms dependent on dsDNA in SLE. Moreover, individuals undergoing chronic haemodialysis, who are thereby exposed to a very large cumulative amount of such DNA, might serve as models for studying its long-term sequelae.