The inhibition of polymorphonuclear leukocyte cytotoxicity by dapsone. A possible mechanism in the treatment of dermatitis herpetiformis.

The effect of the sulfone compound 4,4'-diaminodiphenyl sulfone (dapsone) on normal human polymorphonuclear leukocytes (PMNL) has been investigated in vitro. The drug has a dramatically beneficial effect in dermatitis herpetiformis in which the PMNL and immune complexes has been stressed to be of importance for the development of the skin lesions. Pruritus disappears and the inflammatory eruptions clear within a few days of starting therapy. The effect of dapsone has been evaluated on the different stages of phagocytosis. Using dapsone concentrations (1-30 mug/ml) comparable with those found after therapeutic doses, we have found that the drug interferes primarily with the myeloperoxidase (MPO)-H(2)O(2)-halide-mediated cytotoxic system in the PMNL. No effect was observed on random locomotion, chemotaxis, phagocytic ingestion, oxidative metabolism, or the release of lysosomal enzymes. Kinetic studies in a cell-free system with purified MPO revealed a competitive type of inhibition using varying concentrations of NaI. Furthermore, the inhibition resulted in reduced candidicidal activity during phagocytosis of Candida albicans, and reduced cytotoxicity to adjacent mammalian cells measured as the (51)Cr release from virus-induced lymphoma cells. Because the MPO-H(2)O(2)-halide system not only fulfills the antimicrobial activity but is suggested to be a modulator of the inflammatory reaction as well, the action of dapsone in dermatitis herpetiformis may in part be explained by its effect on this system.

Glutaric acidemia type II. Heterogeneity in beta-oxidation flux, polypeptide synthesis, and complementary DNA mutations in the alpha subunit of electron transfer flavoprotein in eight patients.

We studied metabolic, polypeptide and genetic variation in eight glutaric acidemia type II (GA II) patients with electron transfer flavoprotein (ETF) deficiency. As measured by 3H-fatty acid oxidations in fibroblasts, beta-oxidation pathway flux correlated well with clinical phenotypes. In six patients with severe neonatal onset GA II, oxidation of [9,10(n)-3H]-palmitate ranged from 2% to 22% of control and of [9,10(n)-3H]myristate, from 2% to 26% of control. Of two patients with late onset GA II, one had intermediate residual activities with these substrates and the other normal activities. Radiolabeling and immunoprecipitation studies revealed that three of the six neonatal onset GA II patients had greatly diminished or absent alpha- and beta-ETF subunits, consistent with a failure to assemble a stable heterodimer. Another neonatal onset patient showed normal synthesis of beta-ETF but decreased synthesis of alpha-ETF. Two neonatal onset and two late onset GA II patients showed normal synthesis of both subunits. Analysis of the pre-alpha-ETF coding sequence revealed seven different mutations in the six patients with neonatal onset GA II. The most common mutation was a methionine for threonine substitution at codon 266 found in four unrelated patients, while all the other mutations were seen in single patients. No mutations were detected in the two patients with late onset GA II.

Myeloperoxidase modulates the phagocytic activity of polymorphonuclear neutrophil leukocytes. Studies with cells from a myeloperoxidase-deficient patient.

Patients lacking the primary granulae enzyme, myeloperoxidase (MPO), do not usually show any increased susceptibility to infection or altered inflammatory response, in contrast to several other biochemical defects in polymorphonuclear neutrophils. We have now evaluated the role of MPO on phagocyte function in a patient with complete MPO deficiency suffering from generalized pustular psoriasis. We found that the MPO-deficient neutrophils showed enhanced phagocytosis (greater than 200% of normal) of IgG- and C3b-opsonized yeast particles and prolonged N-formylmethionyl-leucyl-phenylaline-mediated stimulation of superoxide production. When purified human MPO was added to normal neutrophils during cell adhesion, their Fc- and C3b-mediated phagocytosis was reduced without affecting cell viability. 1 microgram/ml of MPO reduced the Fc and C3b phagocytosis to 47 and 65%, respectively, whereas 10 micrograms/ml reduced the activity to 20 and 54%. Both attachment and ingestion were reduced to a similar extent, indicating that MPO affected the receptor function per se. When MPO was added to the hyperactive MPO-deficient cells, phagocytosis was reduced more rapidly. Catalase, azide, and methionine eliminated the inhibitory effect, and catalase and methionine, in fact, enhanced the phagocytic activity of adherent neutrophils. These data indicate that, apart from being a potent antimicrobial system, the oxidizing activity of the MPO-H2O2-halide system may modulate the inflammatory response by impairing certain receptor-mediated recognition mechanisms of phagocytic cells, which otherwise could elicit inflammatory reactions and tissue injury.

Immunodetection of endogenous opioid peptides in human brain tumors and associated cyst fluids.

The antitumorigenic effects of endogenous opioid peptides and their presence in extracerebral tumors are well documented. In this study, methionine-enkephaline (met-enkephalin) was measured by radioimmunoassay in 108 glial and nonglial brain tumors and in 44 associated cyst fluids. By immunohistochemistry, the distribution of the peptide and its precursor, preproenkephalin A, was also analyzed. Met-enkephalin and preproenkephalin were detected in the cytoplasm and cell processes of all tumors. Moreover, for neuroectodermal tumors (i.e., gliomas, gangliogliomas, and dysembryoplastic neuroepithelial tumors), a strong inverse correlation (P < 0.0001) was observed between the met-enkephalin levels and the degree of malignancy (242.9, 148.3, 55.3, and 30.3 pg/mg protein for grade 1, 2, 3, and 4, respectively). When compared to normal tissue, this differential expression mainly results from a decrease in the opioid peptide content in high-grade neuroectodermal tumors. Meningiomas and cerebral metastases displayed low met-enkephalin levels, similar to those of grade 4 neuroectodermal tumors. Large amounts of met-enkephalin were found in all cyst fluids. These data suggest that the endogenous opioid system is an integral component of brain tumors and that met-enkephalin may represent a useful malignancy marker in neuroectodermal tumors.

Complexity of developmental control: analysis of embryonic cell lineage specification in Caenorhabditis elegans using pes-1 as an early marker.

In the early Caenorhabditis elegans embryo five somatic founder cells are born during the first cleavages. The first of these founder cells, named AB, gives rise to 389 of the 558 nuclei present in the hatching larva. Very few genes directly involved in the specification of the AB lineage have been identified so far. Here we describe a screen of a large collection of maternal-effect embryonic lethal mutations for their effect on the early expression of a pes-1::lacZ fusion gene. This fusion gene is expressed in a characteristic pattern in 14 of the 32 AB descendants present shortly after the initiation of gastrulation. Of the 37 mutations in 36 genes suspected to be required specifically during development, 12 alter the expression of the pes-1::lacZ marker construct. The gene expression pattern alterations are of four types: reduction of expression, variable expression, ectopic expression in addition to the normal pattern, and reduction of the normal pattern together with ectopic expression. We estimate that approximately 100 maternal functions are required to establish the pes-1 expression pattern in the early embryo.

Structure and Characterization of Eriphia verrucosa Hemocyanin.

Arthropod hemocyanins (Hcs) are a family of large extracellular oxygen-transporting proteins with high molecular mass and hexameric or multi-hexameric molecular assembly. This study reports for the first time the isolation and characterization of the structure of an arthropod hemocyanin from crab Eriphia verrucosa (EvH) living in the Black Sea. Its oligomeric quaternary structure is based on different arrangements of a basic 6 × 75 kDa hexameric unit, and four of them (EvH1, EvH2, EvH3, and EvH4) were identified using ion-exchange chromatography. Subunit 3 (EvH3) shows high similarity scores (75.0, 87.5, 91.7, and 75.0 %, respectively) by comparison of the N-terminal sequence of subunit 1 from Cancer pagurus of the North Sea (Cp1), subunits 3 and 6 of Cancer magister (Cm3 and Cm6), and subunit 2 of Carcinus aestuarii (CaSS2), respectively. Moreover, a partial cDNA sequence (1309 bp) of E. verrucosa hemocyanin encoding a protein of 435 amino acids was isolated. The deduced amino acid sequence shows a high degree of similarity with subunits 3, 4, 5, and 6 of C. magister (81-84 %). Most of the hemocyanins are glycosylated, and three putative O-linkage sites were identified in the partial amino acid sequence of EvH at positions 444-446, 478-480, and 547-549, respectively. The higher stability of native Hc in comparison to its subunit EvH4 as determined by circular dichroism (CD) could be explained with the formation of a stabilizing quaternary structure.

Met-enkephalin receptors in human gliomas.

Endogenous opioid systems (opioid peptides and receptors) are involved in many functions including the regulation of cell growth. We investigated the presence of Met-enkephalin binding sites in gliomas by displacement assays. Results demonstrated that few gliomas exhibit Met-enkephalin binding sites and that the percentage of tumours which express these binding sites strongly decreases with increasing malignancy. Moreover, we observed a shift from mu Met-enkephalin binding sites in low grade gliomas to delta Met-enkephalin binding sites in high grade gliomas. These results suggest an inactivation of the Met-enkephalinergic system in gliomas which could lead to loss of the inhibitory effect exerted by Met-enkephalin on normal astrocyte growth and thus favour progression of malignancy.

VATS increases costs in patients undergoing lung biopsy for interstitial lung disease.

Proliferation in the use of video-assisted thoracic surgery (VATS) has occurred without data demonstrating benefit. Few studies have critically compared VATS with limited thoracotomy (LT) in homogeneous patient populations undergoing standardized procedures. We retrospectively reviewed the hospital records of 37 consecutive patients referred for elective lung biopsy as part of an ongoing interstitial lung disease protocol to determine whether VATS improved outcome or reduced costs. Sixteen patients underwent VATS, and 21 patients underwent LT lung biopsy over a 31 month period. The two groups were homogeneous in regard to clinical symptoms, radiologic findings, age, sex, and preoperative pulmonary function. The operative mortality was not different between the two groups (VATS, 0/16, and LT, 1/21). The postoperative stay was 4.8 +/- 1.0 days for VATS and 5.0 +/- 0.5 days for LT (p = not significant). Operating time, number of specimens obtained, chest tube output, and day of chest tube removal did not differ. There was no difference in the amount of analgesics required during the postoperative period. Operating room cost for VATS was significantly greater than that for LT ($2,663 +/- $384 versus $1,801 +/- $94; p = 0.04) despite the use of nondisposable equipment. Anesthesia-related costs were also greater for VATS ($309 +/- $11 versus $244 +/- $15; p = 0.002). In conclusion, lung biopsy in patients with interstitial lung disease can be performed safely and efficiently with either VATS or LT, but VATS results in higher procedure-related costs.

Precision of digoxin radioimmunoassays and matrix effects: four kits compared.

We studied the interference of the sample matrix on digoxin radioimmunoassays using four commercial kits. Plasma samples from non-digitalized patients of the following categories were assayed: uncomplicated essential hypertension treated with spironolactone, uremia, and acute myocardial infarction (AMI). Digoxin 2.50 nmol/L was added to all samples. Digoxin in plasma from patients on spironolactone was overestimated by two of the kits (means 2.77 and 2.68 nmol/L, respectively; p less than 0.01) and underestimated in samples from uremic patients by one kit (2.32 nmol/L; p less than 0.01). The digoxin content of AMI plasma was overestimated by one kit (2.62 nmol/L; p less than 0.05). Significant differences were found between radioimmunoassays when estimating digoxin concentration in the same category of patient and within individual methods used for different categories. Precision expressed as 95% confidence intervals ranged from 0.43 to 0.80 nmol/L for the kits. Thus, deviations in recorded digoxin concentrations from the true values found, but were of secondary importance because of the relatively low precision of the assays.

Clofazimine. A new agent for the treatment of pyoderma gangrenosum.

Eight patients had pyoderma gangrenosum. They were given a phendimetrazine tartrate derivative, clofazimine (Lamprene [Britain]), which is a chemotherapeutic agent used mainly in certain mycobacterial infections and which also has phagocytosis-enhancing properties. The effect of this drug was remarkably good, with rapid healing of the lesions commencing 3 to 14 days after treatment was started. The mechanism for the effect of clofazimine in pyoderma gangrenosum is not known.

Comparative in vivo evaluation of a radioimmunoassay and a chromatographic assay for the measurement of digoxin in biological fluids.

The concentrations of digoxin in plasma and urine samples obtained from three healthy male volunteers, who received 1.2 mg of labeled digoxin perorally and intravenously, were simultaneously measured by a commercially available radioimmunoassay (RIA) and by a combined column thin-layer chromatographic assay (CA). The CA method, previously shown to assay digoxin specifically, was also used to monitor the individual digoxin metabolites. The results of this investigation showed that digoxin was significantly metabolized, particularly after peroral administration. The lower level of sensitivity of the RIA in plasma was 0.4 ng/mL. There were highly significant positive linear correlations between the values of the following parameters of digoxin as obtained by the RIA and CA methods: the concentrations in plasma and urine, the AUCs, and the cumulatively excreted amounts in urine. The two assays did not give completely identical results either with plasma or urine; the slopes of the regression lines deviated from unity in a significant number of cases. However, there was no relationship between the magnitude of the slopes of the regression lines and the extent of metabolism. It was concluded that the commercially available RIA evaluated was specific for digoxin and that the presence of digoxin metabolites did not affect the determinations.

Do contraceptives influence the incidence of acute pelvic inflammatory disease in women with gonorrhoea?

The influence of different contraceptive techniques on the incidence of pelvic inflammatory disease (PID) in 672 patients with gonorrhoea have been studied. The lowest frequency of PID was found in patients using hormonal contraceptives (Group A), 8.8 per cent compared to 23.5 per cent in patients using intrauterine devices (Group B) and 15.1 per cent in patients using neither technique (Group C). In comparable control groups no significant differences in background factors, such as age, marital status and sexual activity were demonstrated. It is therefore concluded that the significantly lower incidence of PID in patients using hormonal contraceptives compared to the other groups and the high incidence of PID in patients using intrauterine devices is related to the contraceptive technique per se.

Problems with the immunoassay of digoxin.

Factors that affect the standardization and reliability of the radioimmunoassay of digoxin are reviewed. Some new data are presented on standardization and suggestions are made for dealing with problems in the design and techniques of assays.

Discoid lupus erythematosus treated with cryotherapy.

A female is presented with multiple longstanding hypertrophic discoid lupus erythematosus lesions resistant to topical and systemic therapy for years. She suffered severe itching and pain localized to the lesions. Treatment with cryosurgery resulted in complete healing of the lesions, leaving slightly hypopigmented soft scars. There was no tendency of relapse during a follow-up period of ten years.

Determination of ethylene glycol in postmortem blood by capillary gas chromatography.

A simple procedure for the determination of ethylene glycol in blood by capillary gas chromatography was developed. The proteins are precipitated by the addition of perchloric acid which includes the internal standard 1,2-butanediol. The extract is neutralized and the solution is directly injected. The assay is linear and the precision, expressed as coefficient of variation, is 4-11% (within run). The detection limit is about 0.05 g/L. The method also seems applicable for the determination of ethylene glycol in urine.

First observations on high-dosed and long-term thymopentin treatment in active rheumatoid arthritis.

In this pilot study carried out in two centres, six male and two female patients with severe active rheumatoid arthritis (RA) (average duration over 10 years) were treated with thymopentin 50 mg in the form of prolonged i.v. injection (over 10 min), 3 times weekly for 3 to 20 weeks. Two of these patients were subsequently treated with different s.c. doses of thymopentin in a crossover fashion for more than two years, including periods without any treatment or treatment with placebo. The overall clinical efficacy was judged by assessing pain patterns and joint status and the functional stage of the patients according to Steinbrocker; in addition, the sedimentation rate was measured before and after the therapy. Seven out of eight patients showed definite improvement in their clinical status as assessed by the Steinbrocker scale. Most of the symptoms, particularly pain, capsular swelling, tenderness and morning stiffness, were remarkably reduced within 3 weeks of thymopentin treatment. Sedimentation rate decreased in five out of eight patients. Prolonged i.v. injections seemed to have somewhat better effects than s.c. administration; in the latter group the highest dose (3 X 100 mg/week or higher) produced the best results. During placebo treatment and during the medication-free intervals both groups of patients got worse. No side-effects occurred during the study.

The effect of thymopentin treatment on the relapse rate in frequently relapsing herpes simplex virus infections.

Twenty-four patients suffering from longstanding severe recurrent herpes simplex, who had not responded to prior therapy, were treated with s.c. thymopentin injections 50 mg, three times weekly, over a period of six weeks. They were followed up at weekly intervals over this period and then six weeks later. Moreover, the longest relapse-free period observed in the year after the treatment was recorded in the investigator's documentation. Thirteen of the 14 patients with labial herpes simplex and 10 of the 13 patients with genital herpes simplex improved markedly as shown by a decrease in the relapse rate of at least 50%, shorter episodes of relapse and improvement of symptoms such as pain and itching. Fourteen of these 27 patients experienced no relapse for a period longer than four months after cessation of the therapy. No serious side-effects were observed. Laboratory examinations before, during and after thymopentin did not reveal significant alterations except for an increase in the T-helper/T-suppressor ratio. The effect of thymopentin is assumed to be due to T-helper cell activation resulting in enhanced interleukin-2 production with subsequent proliferation of cytotoxic T lymphocytes and natural killer cells which are capable of producing immune interferon.

Aspects of the treatment of mycosis fungoides. A report from the Scandinavian Mycosis Fungoides Study Group.

The Scandinavian Mycosis Fungoides Study Group includes dermatologic clinics in Denmark, Norway, and Sweden. The results of the first three years of activity are presented herein. In plaque stage, topical nitrogen mustard was highly effective. Preceding intravenous tolerance induction seems to be of no value. PUVA induced equally high remission rates. Both modalities were also highly effective in cutaneous tumor stage. In advanced tumor stage and in case of extracutaneous involvement systemic chemotherapy was given. Topical treatment alone was more effective on the cutaneous lesions including tumors than systemic chemotherapy alone. Therefore a combination of topical and systemic treatment is recommended in advanced stages of mycosis fungoides.

Multivariate analysis approach to the serum peptide profile of morbidly obese patients.

BACKGROUND: Obesity is currently epidemic in many countries worldwide and is strongly related to diabetes and cardiovascular disease. Mass spectrometry, in particular matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) is currently used for detecting different pattern of expressed protein. This study investigated the differences in low molecular weight (LMW) peptide profiles between obese and normal-weight subjects in combination with multivariate statistical analysis. MATERIALS: Serum samples of 60 obese patients and 10 healthy subjects were treated by cut-off membrane (30000 Da) to remove the most abundant proteins. The filtrates containing the LMW protein/peptides were analyzed by MALDI-TOF mass spectrometry. Dataset was elaborated to align and normalize the spectra. We performed cluster analysis and principal component analysis to detect some ionic species that could characterize and classify the subject groups. RESULTS: We observed a down-expression of ionic species at m/z 655.94 and an over-expression of species at m/z 1518.78, 1536.77, 1537.78 and 1537.81 in obese patients. Furthermore we found some ionic species that can distinguish obese patients with diabetes from those with normal glucose level. CONCLUSION: Serum peptide profile of LMW associate with multivariate statistical approach was revealed as a promising tool to discriminate and characterize obese patients and it was able to stratify them in relation to comorbidity that usually are associated with this disease. Further research involving a larger sample will be required to validate these findings.

A comparison between MALDI-MS and CE-MS data for biomarker assessment in chronic kidney diseases.

Non-invasive detection of diseases, based on urinary proteomics, is becoming an increasingly important area of research, especially in the area of chronic kidney disease (CKD). Different platforms have been used in independent studies, mostly capillary-electrophoresis coupled ESI-MS (CE-MS), liquid chromatography coupled mass spectrometry, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We have compared the performance of CE-MS with MALDI-MS in detecting CKD, based on a cohort of 137 urine samples (62 cases and 75 controls). Data cross-talk between the two platforms was established for the comparison of detected biomarkers. The results demonstrate superior performance of the CE-MS approach in terms of peptide resolution and obtained disease prediction accuracy rates. However, the data also demonstrate the ability of the MALDI-MS approach to separate CKD patients from controls, at slightly reduced accuracy, but expected reduced cost and time. As a consequence, a practical approach can be foreseen where MALDI-MS is employed as an inexpensive, fast, and robust screening tool to detect probable CKD. In a second step, high resolution CE-MS could be used in those patients only that scored negative for CKD in the MALDI-MS analysis, reducing costs and time of such a program.