Diurnal variation of steroid hormones and their reference intervals using mass spectrometric analysis.

OBJECTIVE: Accurate measurement of steroid hormones remains challenging. Mass spectrometry affords a reliable means for quantitating steroid profiles accurately. Our objective was to establish and define (1) the extent of diurnal fluctuations in steroid concentrations that potentially necessitate strict adherence to time of sample acquisition and (2) time-dependent steroid reference intervals. DESIGN: Nine steroid markers were examined in couplets in males and females. METHODS: Using isotope dilution high-performance liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis, we developed a multi-steroid profile requiring only a minimal volume of serum (0.1 mL). Couplet (AM and PM) measurements of steroid hormones for 120 healthy females (F) and 62 healthy males (M) were obtained. Patients were recruited from several participating centers. RESULTS: The following diurnal values were noted to be significantly different in both females and males: cortisone, cortisol, corticosterone, 11 deoxycortisol (11 DOC), androstenedione, 17a-hydroxyprogesterone (17 OHP) and dehydroepiandrosterone (DHEA). Testosterone was only found to have significant diurnal variance in males. Progesterone showed no significant difference in AM and PM values for either groups and thus may provide an internal control. CONCLUSIONS: When diagnosing endocrine disorders, it is imperative to acknowledge the 24-h diurnal variation of the biochemical steroid markers. We highlight the importance of standardization of collection times and appropriate implementation of reference intervals. PRECIS: We identify diurnal fluctuations in steroid concentrations with time of day and emphasize the importance of adhering to firm time of sample acquisition.

Does TSH Reliably Detect Hypothyroid Patients?

OBJECTIVES: To evaluate the reliability of normal Thyroid Stimulating Hormone (TSH) as a thyroid function test and assess the effect of Adrenocorticotropic Hormone (ACTH) on serum TSH concentration. DESIGN AND METHODS: Patients presenting to the National Institutes of Health Department of Endocrinology outpatient clinic with symptoms consistent with hypothyroidism were identified. Thyroid hormone concentrations were measured by liquid chromatography/tandem mass spectrometry and immunoassay. Patients with normal TSH concentrations were assessed for both clinical and biochemical hypothyroidism.We evaluated the effect of ACTH stimulation (performed on patients for assessment of adrenal function) on TSH concentration. RESULTS: Patients with symptoms consistent with hypothyroidism but with normal TSH values in the range of 1-4 IU/mL and normal free T4 (FT4) values by immunoassay measurements were confirmed to be biochemically hypothyroid following measurements of thyroid hormones by mass spectrometry. We present case studies of two patients, a 76-year-old male and a 58-year-old female. Improvement in the male patient's hypothyroid symptoms, including afternoon fatigue, constipation, alopecia, dry skin and high cholesterol, was documented after initiating thyroid hormone replacement.ACTH stimulation resulted in an average decrease of 17% in TSH between time 0 and 60 minutes post stimulation. CONCLUSION: Although measurement of TSH is a convenient screen for thyroid function, it is influenced by many factors which may affect its overall reliability. We believe thyroid function should be assessed by more than a single test. We recommend measurement of thyroid hormone concentrations by mass spectrometry if the patient's clinical presentation is discordant with their TSH levels.

[Management of a 4MRGN Acinetobacter baumanii outbreak in a burn unit]. / Management eines Ausbruches mit 4 MRGN Acinetobacter baumannii in einem Brandverletzten-zentrum.

Patients with 4MRGN Acinetobacter baumanii infections in a burn unit represent great challenge. The structured management with 7 involved patients in such a situation is presented. After discovering the infectious trigger a management team is established. An immediate stop for further admissions was announced and all infected room areas and medical equipment were analysed for infection foci. The infected patients were transferred to regional hospitals or a rehabiltation hospital after finishing all surgical procedures. In one case, for whom further operations were needed, a transfer to a separated area of the intermediate care unit (IMC) within the hospital was arranged. The performed analysis of infection foci indicated a bronchoscopy tower to be the infection source. The outbreak was terminated after transferring all patients, final disinfection and subsequent nebulisation with 5-6% hydrogen peroxide within 18 days.

Different binding mechanisms of myeloid leukemic cells to adhesion molecules on bone marrow stromal fibroblasts induced by TNF-alpha and IL-4.

To study the mechanisms of adhesion of myeloid leukemic cells to bone marrow stroma, we analyzed the interaction of bone marrow stromal fibroblasts with myeloid leukemic cell lines and the modulation of adhesion molecule expression on stromal fibroblasts by TNF-alpha and IL-4. Like others, we found up-regulation of VCAM-1 and ICAM-1 on fibroblasts with TNF-alpha treatment, whereby IL-4 acted synergistically with TNF-alpha. VCAM-1 expression on the cell surface was maximal after 10 h, while ICAM-1 expression increased up to 48 h. All myeloid leukemic cell lines tested (HL-60, K562, TMM, U937, ML-2, PLB-985, THP-1, KG1a) revealed weak adhesion to untreated bone marrow fibroblasts (< or =10% bound cells). TNF-alpha and IL-4 significantly enhanced adhesiveness of fibroblasts to the cell lines PLB-985, THP-1, and ML-2, with a peak between 6 and 10 h of treatment. Adhesiveness to the cell line TMM was increased up to eightfold in a time-dependent manner for up to 48 h. The enhanced binding of ML-2-, THP-1-, and PLB-985 cells to stimulated fibroblasts was due at least partially to the interaction of VLA-4 with VCAM-1. Increased adhesion of TMM cells was impaired neither by antibodies to VLA-4, LFA-1, or Mac-1 nor by antibodies to their counter-receptors VCAM-1 or ICAM-1, suggesting that adhesion molecules distinct from VCAM-1 or ICAM-1 are involved in enhanced adhesiveness of the fibroblasts to myeloid leukemic cells.

Effects of bone marrow fibroblasts on the proliferation and differentiation of myeloid leukemic cell lines.

The effects of normal bone marrow fibroblasts (BM FB) on proliferation and differentiation of 10 myeloid leukemic cell lines were investigated in a serum-free co-culture system. The proliferation of three of the cell lines was supported by BM FB. Three of the myeloid cell lines were inhibited 40-70%. The co-culture supernatants were tested for the secretion of hematopoietic cytokines by bioassays. Except for IL-6, which was already produced constitutively by BM FB, only little amounts of interleukin-1 (IL-1), granulocyte colony-stimulating factor (G-CSF), or granulocyte-macrophage colony-stimulating factor (GM-CSF) could be detected in several co-culture supernatants. It could be shown that, according to cytologic and functional criteria, the myeloid leukemic cell lines ML-2 and PLB-985 differentiate along the monocyte-macrophage pathway after co-culture with BM FB. They revealed a histiocytic phenotype and could be induced to produce reactive oxygen intermediates (ROI) after stimulation with zymosan or phorbol-myristate-acetate (PMA). Additional proof for differentiation was obtained from flow cytometric analysis of surface differentiation antigens and adhesion molecules. The neutralization of IL-6 activity in the co-cultures by antibodies resulted in prevention of differentiation of PLB-985 cells, while differentiation of ML-2 cells in the co-cultures was not affected by addition of anti-IL-6 antibodies. Furthermore, in co-culture experiments with fibroblasts from skin and foreskin, we found a differentiation of PLB-985 cells comparable to that in co-cultures with BM FB, but poor differentiation of ML-2 cells. These data suggest that different mechanisms are involved in the differentiation of ML-2 and PLB-985 cells.

Tumorigenicity, mucin production and AM-3 epitope expression in clones selected from the HT-29 colon carcinoma cell line.

Two mucin-producing cell clones (16.2 and 12.2) and a mucin-deficient clone (15.2) were selected from the established human adenocarcinoma cell line HT-29 by limiting dilution and Alcian blue staining. The amounts of the mucin antigen detectable on the cell surface with the monoclonal antibody (MAb) AM-3 decreased in the order HT-29 greater than 16.2 greater than 12.2 greater than 15.2 = 0. The binding avidity of AM-3 antibody to cells as well as to mucin extracts from each cell line decreased in the same order, indicating that the epitope density on the cell-bound mucins was highest in HT-29 and lowest in 12.2 cells. The parental line and the mucin-producing cell clones 16.2 and 12.2 showed no contact inhibition and grew as aggregates, while the 15.2 cells were well spread and formed a regular monolayer. The mucin-producing cell lines injected into nude mice yielded solid tumors with different growth rates (HT-29 greater than 16.2 greater than 12.2), while the 15.2 cell clone was not tumorigenic at all. The relative amounts of total mucin-bound hexoses and of the mucin epitope AM-3 decreased in the xenografts in the order HT-29 greater than 16.2 greater than 12.2. The present system is suitable for investigating the role of mucins in growth of colon carcinoma cells and indicates that increased tumorigenicity in nude mice coincides with the increase in total mucin expression and the expression of the AM-3 mucin epitope in tumor tissue.

Increased number of accessible sugar epitopes defined with monoclonal antibody AM-3 on colonic mucins is associated with malignant transformation of colonic mucosa.

Monoclonal antibody AM-3 detects a mucin sugar epitope (AM-3 epitope) the expression of which increases in the course of human colon carcinogenesis parallel to the gradual morphological alterations (so called adenoma-carcinoma sequence). In the present report the AM-3-positive mucin has been purified from human normal and carcinomatous colonic tissue. About 300-fold enrichment of the epitope per protein from both sources was achieved after ultracentrifugation, gel filtration on Sepharose CL-6B and isopyknic gradient centrifugation. Slot-blot and enzyme-linked immunosorbent assays of the purified preparation indicated not only different amounts of the mucins but also a consistent qualitative difference between the molecules from both sources. The qualitative difference could be obliterated by a partial removal of the AM-3 epitope from the tumor-derived mucin with neuraminidase. The visualization of the molecules by rotary shadowing indicated that the mucins from both sources have similar length distribution, 80% of the molecules being 100-600 nm long. The reaction with AM-3 antibody followed by rotary shadowing showed that in the purified preparations more than 95% of the tumor-derived molecules and 74% of the normal colon tissue-derived molecules carried the epitope. The tumor-derived mucins bound, on the average, 34 +/- 15 (SD) antibodies/1000 nm of the protein core while the mucin from normal colon tissue carried 12 +/- 11 antibodies/1000 nm of the protein core. These data indicate that the increased expression of AM-3 epitopes during malignant transformation of the human colon is due to accumulation of AM-3-positive mucin as well as a higher number of accessible AM-3 epitopes on this mucin.

Antigen distribution in organs of mink with Aleutian disease parvovirus infection.

On tissues from naturally infected non-Aleutian mink an immunohistological study was performed using monoclonal antibodies and the immunoperoxidase method. Structural proteins of ADV were demonstrated in cryosections and in ethanol-fixed and paraffin-embedded material which provide antigen detection in a similar amount together with good histological structure. In lymphoid organs viral antigen was restricted to B-cell areas, particularly lymphoid follicles. The pattern of antigen distribution was typical for follicular dendritic cells which are capable to retain immune complexes. Beside macrophages in the interior of lymphoid follicles most likely proliferating B-lymphoblasts reveal nuclear and cytoplasmatic presence of structural proteins indicating viral replication. Cells of the mononuclear phagocyte system such as cells of lymphatic sinuses and hepatic Kupffer cells harbor viral protein in the cytoplasm, probably resulting from phagocytosis of immune complexes. Renal glomeruli were consistently negative for virus antigen whereas in interstitial infiltrates cells resembling macrophages stained positive for ADV structural proteins.

Violet mink develop an acute disease after experimental infection with Aleutian disease virus (ADV) isolate ADV SL3.

Six-Aleutian (aa)-genotype violet mink were infected intraperitoneally with the Aleutian Disease Virus (ADV) bone marrow derived isolate ADV SL3. All animals developed virus-specific antibodies and hypergammaglobulinaemia. Mortality during the fourteen week duration of the infection was 50%. The virus induced (histo)pathological lesions typical for Aleutian Disease. By immunohistochemical examination using a virus capsid-specific monoclonal antibody viral antigen was detected in lymph nodes, spleen, kidneys and once in hepatic Kupffer cells. By Southern blot and in situ hybridization studies with strand-specific RNA probes able to distinguish viral replicative forms from merely sequestered genomic DNA, ADV replication was detected in mesenteric lymph nodes and spleen. In one mink DNA replicative forms were also found in bone marrow cells or mononuclear cells of the peripheral blood, respectively. Only single-stranded viral DNA was detected in liver, kidney, gut and lung of infected animals. From Southern blot hybridization results a different, possibly organ-specific permissiveness of ADV in vivo is suggested.

Mechanisms contributing to the virus persistence in Aleutian disease.

In this review published results and further studies concerning the persistence of Aleutian disease virus (ADV) isolate SL3 are presented. By Southern blot and in situ hybridization with strand-specific RNA probes focal replication of ADV-DNA was demonstrated in spleen, mesenteric lymph nodes, sporadically in mononuclear cells of the peripheral blood and bone marrow cells. These findings further support the concept of the lymphotropism of ADV. All cell culture-adapted ADV strains appear to have a ts-defect. Our in vitro studies indicate that the ADV isolate G(orham) induced the synthesis of comparable amounts of viral replicative DNA and viral proteins VP1 and VP2 at the non-permissive temperature of 37 degrees C. However, the viral progeny DNA synthesis was about threefold less at 37 degrees C compared to the permissive temperature of 32 degrees C. These findings suggest that the reduced level of viral progeny DNA at 37 degrees C accounts for the reduced production of infectious ADV. Finally, we provided experimental evidence that the apparent lack of neutralizing antibodies in AD is due to the masking of critical viral epitopes by cellular phospholipids.

In vitro assay to test differential substrate affinities of growing axons and migratory cells.

An in vitro assay is presented in which different soluble substrates are arranged in narrow alternating stripes which forces growing axons and migratory cells to choose between them. The usefulness of this assay is exemplified by offering goldfish retinal axons and glial cells of the optic nerve a variety of substrates in stripes. Given a choice between substrates of unequal growth supporting activities axons and migratory cells grow in stripes, thus expressing their preference for one of the substrates. Growth in stripes was observed 1. when a substrate with growth promoting properties was next to one which did not possess these properties, 2. when the growth promoting activity of a substrate applied to both stripes was in one stripe blocked by an antibody, 3. when two different growth promoting substrates were offered.

Efficient medium for impingement and storage of enveloped viruses.

Airborne infections with pathogenic viruses play an important role in the transmission of diseases amongst men and animals. We compared several media intended for impingement of viruses from virus-contaminated air and for their preserving effect for two enveloped viruses. Sindbis (SINV) and vesicular stomatitis virus (VSV), members of the families Toga- and Rhabdoviridae, respectively, were chosen as indicator agents. Amongst the media tested, a sampling fluid consisting of phosphate buffered saline, pH 7.2, 0.5% bovine serum albumin, 0.5% gelatine (PBSplus) was most efficient to minimize the sampling stress during impingement and to preserve the infectivity SINV and VSV under stringent conditions at 37 degrees C. About 50% of virus infectivity was recovered 15.7 or 30 hours, respectively, after the beginning of storage. Thus the recommended medium is also suitable for shipment and storage of diagnostic virus samples.

Recognition of position-specific properties of tectal cell membranes by retinal axons in vitro.

In order to test the preference of growing axons for membrane-associated positional specificity a new in vitro assay was developed. In this assay, membrane fragments of two different sources are arranged as a carpet of very narrow alternating strips. Axons growing on such striped carpets are simultaneously confronted with the two substrates at the stripe borders. If there is a preference of axons for one or the other substrate they become oriented by the stripes and grow within the lanes of the preferred substrate. Such preferential growth could, in principle, be due to affinity to attractive factors on the preferred stripes or avoidance of repulsive factors on the alternate stripes. This assay system was used to investigate growth of chick retinal axons on tectal membranes. Tissue strips cut from various areas of the retina were explanted and the extending axons were confronted with stripes of cell membranes from various areas within the optic tectum. Tectal cell membranes prove to be an excellent substrate for the growth of retinal axons. Nasal and temporal axons can grow well on membranes of both posterior and anterior tectal cells. If, however, temporal axons are given a choice and encounter the border between anterior and posterior membranes they show a marked preference for growth on membranes of the anterior tectum, their natural target area. Nasal axons do not show a preference in this assay system. The transition from nasal to temporal properties within the retina is abrupt. In contrast, the transition from anterior to posterior properties of the tectal cell membranes occurs as a smooth gradient. Significantly, the positional differences of tectal membrane properties are only seen during the period of development of the retinotectal projection and are independent of tectal innervation by retinal axons. These anterior-posterior differences disappear by embryonic day 14.

Apparent lack of neutralizing antibodies in Aleutian disease is due to masking of antigenic sites by phospholipids.

It is generally accepted that Aleutian disease virus (ADV) cannot be neutralized by antibodies either in vivo or in vitro. We found several ways to demonstrate neutralization of ADV by specific antibodies from mink. It was essential to make ADV monodisperse by treatment with sodium lauroyl sarkosyl or n-butanol or by filtration through 0.05-micron membranes before neutralization tests. In kinetic experiments, there was a 95% loss of virus infectivity within the first 5 min of reaction, but a resistant fraction of about 1% remained after 1.5 hr of incubation. Neutralization titers between 1:160 and 1:640 were found in sera from naturally and experimentally infected mink. A positive relation was consistently found between neutralization and ELISA titers. Furthermore, separation of phospholipids from ADV was shown by thin-layer chromatography of butanol-extracted virions. By reconstitution of monodispersed ADV with various lipids, phospholipids were found to interfere with virus neutralization by attachment to the virus surface.

Epitopic mapping of structural and nonstructural Aleutian disease virus proteins.

Common antigenic properties for p85 and p75 but a different antigenic character for p71 Aleutian disease virus (ADV) proteins were demonstrated by Western blot analysis with monoclonal antibodies. It was shown that four hybridomas (ADV-Hy 47, 66, 77 and 84) with specific reactivity for structural proteins p85 and p75 also recognized p25 but not the p71, nonstructural, protein. In turn, the monoclonal antibody ADV-Hy 2 recognized the p71 protein only. For further studies of their antigenic properties, the ADV proteins were subjected to enzymatic or chemical cleavage. The derived peptide fragments were analyzed by epitopic mapping. Depending on the cleavage reagent and monoclonal antibody applied, specific peptide maps were revealed. The maps of p85 and p75 were very similar, indicating that both proteins shared an extensive antigenic relationship. After cleavage with alpha-chymotrypsin and N-chlorosuccinimide and by using the ADV-Hy 84 monoclonal antibody, unique peptide fragments were identified with p85 which had no counterparts in p75 fragments.