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The erythroleukemia cell lines K562 and human erythroleukemia (HEL) are established models to study erythroid and megakaryocytic differentiation in vitro. In this study, we show that the alpha1-adrenergic antagonists, benoxathian and prazosin, inhibit the proliferation and induce apoptosis in K562 and HEL cells. Furthermore, both tested substances induced the expression of the megakaryocytic marker CD41a, whereas the expression of the erythroid marker glycophorin-a was decreased or unchanged. Even though the expression of differentiation markers was similar after benoxathian and prazosin treatment in both cell lines, endomitosis of erythroleukemia cells was observed only after prazosin treatment. So far, benoxathian and prazosin are the first described extracellular ligands, which cause megakaryocytic differentiation in K562 and HEL cells. In summary, these results indicate a possible role of alpha1-adrenergic receptor signaling in the regulation of erythroid and megakaryocytic differentiation, even though the receptor dependence of the observed effects needs further investigation.

The aim of this study was to investigate whether subcutaneous adipose tissue topography (SAT-Top) is different in female CHD patients (n=26) and healthy controls (n=36) matched to age, body size, weight, and BMI. The thicknesses of SAT layers were measured by LIPOMETER at 15 specified body sites. To calculate the power of the different body sites to discriminate between CHD women and healthy controls, receiver operating characteristic (ROC) curve analysis was performed. For each parameter, sensitivity and specificity were calculated at different cutoff points. CHD women showed a significant decrease to 78.36% (p=0.012) at body site 11-front thigh, 73.10% (p=0.012) at 12-lateral thigh, 72.20% (p=0.009) at 13-rear thigh, 66.43% (p<0.001) at 14-inner thigh, and 49.19% (p<0.001) at 15-calf. The best discriminators analysed by ROC curves between female CHD patients and healthy controls turned out to be calf and inner thigh (optimal cut off values: calf: 3.85 mm and inner thigh: 11.15 mm). Stepwise discriminant analysis identified the body sites calf, lateral chest, and inner thigh as significant. In conclusion, information was obtained on the extent to which SAT thickness at each measured body site is able to discriminate between the two subject groups. The good discrimination results obtained for the present dataset are encouraging enough to recommend applying LIPOMETER SAT-Top measurements in further studies to investigate individual risks for CHD.

Objective: Physical cues play a crucial role in skeletogenesis and osteochondral regeneration. Although human mesenchymal stem cells (hMSCs) offer considerable therapeutic potential, little is known about the molecular mechanisms that control their differentiation. We hypothesized that mechanical strain might be an inherent stimulus for chondrogenic and/or osteogenic differentiation in undifferentiated hMSCs, where c-Fos (FOS) might play a major role in mechanotransduction., Method: hMSCs from 10 donors were intermittently stimulated by cyclic tensile strain (CTS) at 3000 mustrain for a period of 3 days. Differential gene expression of strained and unstrained hMSCs was analysed by real-time RT-PCR for several marker genes, including the transcription factors FOS, RUNX2, SOX9, and others. Additionally, alkaline phosphatase activity (ALP) was determined kinetically., Results: The application of CTS significantly stimulated the expression levels of the early chondrogenic and osteogenic marker genes (SOX9, LUM, DCN; RUNX2, SPARC, SPP1, ALPL); this was accompanied by stimulation of ALP activity (+38%+/-12 standard error of mean, P<0.05). Matrix analysis revealed that the osteo-chondrogenic response followed a coordinated expression pattern, in which FOS was attributed to early osteogenic but not chondrogenic differentiation., Conclusion: Undifferentiated hMSCs are highly sensitive to mechanical strain with a transcriptionally controlled osteo-chondrogenic differentiation response in vitro.

Adding the natural antioxidant alpha-tocopherol to ultra-high molecular weight polyethylene (UHMW-PE) can remarkably delay the oxidation of hip cups made thereof. However, alpha-tocopherol is likely to undergo different chemical transformations during manufacturing and sterilization of hip cups than in human metabolism. Therefore, the biocompatibility of the putative transformation products has to be investigated. In-vitro tests with L929 mice fibroblast-cells gave no evidence for cytotoxicity. To further ensure the biocompatibility, in-vitro tests with human cells were carried out in this study. Two different human cell lines, one adherent cell line, HF-SAR, and one suspension culture, GSJO, were tested on UHMW-PE-tablets (diameter: 15 mm; thickness: 2 mm; processed according to standard procedures for artificial hip-cups) with and without alpha-tocopherol with respect to cell viability, proliferation and morphology by means of cell counting, WSt-1 proliferation assay and scanning electron microscopy. Similar proliferation rates were found with both polyethylene samples. Further, we found intact morphology in light and electron microscopy on each substrate. The morphologic characteristics of skin fibroblasts were not changed by any material. Normal adherence and spreading of the fibroblasts was found on controls of glass, as well as on polystyrene and on stabilized and unstabilized polyethylene. The characteristic behaviour as suspension of the GSJO cells remained unchanged. The mitochondrial activity, as studied by WST-1 cell proliferation reagent, was identical on each substrate during the whole observation period of 7 days.

The antioxidant activity of melatonin (MEL) has been considered to constitute part of its physiological as well as pharmacological effects. However, as described herein we found a profound prooxidant activity of micro- to millimolar concentrations of MEL in the human leukemic Jurkat cell line. This prooxidant effect was increased in glutathione-depleted cells and counteracted by antioxidants. As a consequence MEL promoted fas-induced cell death. These data therefore indicate that MEL may be a modulator of the cellular redox status, but does not necessarily act as an intracellular antioxidant.

Aims: Decreased night-time plasma levels of melatonin were recently reported in patients with coronary artery disease, and it was postulated that melatonin production may be impaired, due to a lack of synthesizing enzymes. However, since artefacts possibly influencing the release pattern were not taken into account, this interpretation was strongly criticized. We therefore carefully investigated night-time melatonin production in patients with coronary artery disease using an appropriate experimental approach. Furthermore, we examined the effect of beta-blockers, a frequently used drug in coronary artery disease therapy., Methods and Results: Forty-eight male patients with angiographically documented severe coronary artery disease, 24 of them taking beta-blockers daily in therapeutic dosages, were included. Eighteen age-matched men, with no evidence of coronary sclerosis, served as controls. To determine melatonin production, 6-sulfatoxymelatonin (aMT6s) was measured radioimmunologically from overnight urine. Urinary aMT6s concentration was significantly decreased in patients, and beta-blocker treatment did not further suppress melatonin production., Conclusions: The data obtained using this investigative approach provide clearcut evidence that melatonin production in patients with coronary artery disease is decreased. Whether a decreased melatonin level may be a predisposing factor for coronary artery disease, or whether the occurrence of coronary artery disease decreases melatonin synthesis remains to be determined., (Copyright 1999 The European Society of Cardiology.)

Both melatonin and its precursor N-acetylserotonin have been reported to exert antioxidant properties both in vitro and in vivo. Since little is known about their antioxidant activity in lymphocytes, we investigated their effects on spontaneous and on oxidant-induced reactive oxygen species formation in human peripheral blood lymphocytes in comparison to the antioxidant trolox, a water-soluble analogue of alpha-tocopherol. Both melatonin and N-acetylserotonin exhibited antioxidant properties against t-butylated hydroperoxide- and diamide-induced reactive oxygen species formation in peripheral blood lymphocytes. N-acetylserotonin turned out to be about three times more effective than melatonin. In resting cells, the intracellular reactive oxygen species concentration was only decreased by N-acetylserotonin and trolox, melatonin had no effect. In t-butylated hydroperoxide-mediated cell death, N-acetylserotonin was as effective as trolox in protecting peripheral blood lymphocytes from cell death and required 10-fold lower concentrations than melatonin. Furthermore, in an aqueous cell-free solution, the capacity of N-acetylserotonin to scavenge peroxyl radicals was much higher than that of melatonin. These results clearly indicate N-acetylserotonin to be a much better antioxidant than melatonin.

A significant decrease in %IgG1 accompanied by an increase in %IgG2 in total serum IgG has been previously reported as a highly sensitive marker for detecting early stages of carcinomas of various localizations. Here we investigated the question as to whether this phenomenon is also observed in sera of patients with squamous cell carcinoma of the head-neck region (SCC-HN), and to evaluate its diagnostic performance in the post-operative monitoring. Using quantitative affinity chromatography, serum concentrations of IgG1, IgG2 and total IgG were determined in 81 patients with different stages of primary and untreated SCC-HN, in 51 SCC-HN patients in post-therapeutical follow up, and in 33 patients with organ matched benign diseases. The data were compared with a total of 174 healthy controls. It was found that (i) 105 SCC-HN patients exhibited a mean value of 56.0 +/- 0.7% IgG1, which likewise differed from healthy controls (63.2 +/- 0.5) and benign diseases (61.5 +/- 1.0) with P < 0.0005, (ii) sensitivities and specificities for discriminating primary malignancies from healthy controls were 70 and 74% respectively, and from benign diseases 65 and 76%, (iii) highest sensitivities and specificities were observed with post-therapeutic cases suffering from tumour recurrence (88% and 75%) or patients with distant metastases (87% and 86%), (iv) apparently tumour-free post-therapeutic patients showed a mean %IgG1 not different from the normal value. The decrease in %IgG1 accompanied by increased %IgG2 is an efficient, sensitive and early marker of SCC-HN, which appears particularly useful for the post-therapeutic monitoring.

Circadian alterations of several immune functions in vivo are well established, and may have important physiological and clinical implications. In line with this, tumor necrosis factor (TNF)-alpha has been implicated in the circadian regulation of body temperature. As soluble TNF receptors (TNF-R) act as naturally occurring competitive inhibitors of this cytokine, we investigated plasma levels of the soluble sTNF-R55 and sTNF-R75 in comparison with plasma cortisol and body temperature in nine healthy male volunteers during a defined 16 h light/8 h dark cycle. It was found that sTNF-R75, but not sTNF-R55, exhibited a clear-cut circadian rhythm with a significant (P < 0.01) peak at 7:51 a.m. +/- 58 min. The phase of the sTNF-R75 rhythm preceded that of cortisol by approximately 1 h and inversely corresponded to the circadian rhythm of body temperature. Moreover, the individual amplitudes of sTNF-R75 and body temperature exhibited a significant (P < 0.01) positive correlation. These results suggest that (i) the two sTNF-R are regulated independently, (ii) the sTNF-R75 rhythm is not primarily due to the cortisol rhythm and (iii) the fluctuation of the sTNF-R may contribute to the regulation of body temperature by modulating the availability of free TNF.

Juvenile rheumatoid arthritis (JRA), unlike rheumatoid arthritis of adulthood (RA), is a heterogenous disease comprising at least five subtypes that differ in clinical course and prognosis, and require different therapeutical approaches. As compared to RA, the production of local and systemic cytokines in JRA have not yet been as extensively investigated. In this article we review the available literature on cytokine expression in serum and synovial fluid in all five different subtypes of JRA. Even though the data are still fragmentary, the evidence so far suggests that the determination of serum cytokines yields relevant information as to clinical subtype and inflammatory activity of the disease. Furthermore, the cytokine data suggest that the pathogenesis of JRA may even by more heterogenous than defined by the clinical subtypes. Finally, future directions of research in this area are proposed, and-based on the latest results-arguments for (anti)cytokine therapies in JRA are critically discussed., (Copyright 1998 Academic Press Limited.)

For several years, our group has been studying the in vivo role of adrenergic and cholinergic mechanisms in the immune-neuroendocrine dialogue in the rat model. The main results of these studies can be summarized as follows: (1) exogenous or endogenous catecholamines suppress PBL functions through alpha-2-receptor-mediated mechanisms, lymphocytes of the spleen are resistant to adrenergic in vivo stimulation, (2) direct or indirect cholinergic treatment leads to enhanced ex vivo functions of splenic and thymic lymphocytes leaving PBL unaffected, (3) cholinergic pathways play a critical role in the "talking back" of the immune system to the brain, (4) acetylcholine inhibits apoptosis of thymocytes possibly via direct effects on thymic epithelial cells, and may thereby influence T-cell maturation, (5) lymphocytes of the various immunological compartments were found to be equipped with the key enzymes for the synthesis of both acetylcholine and norepinephrine, and to secrete these neurotransmitters in culture supernatants.

The antioxidant effect of melatonin on LDL oxidation was studied in vitro using either a thermolabile initiator or copper ions to induce lipid peroxidation. Loading of LDL with melatonin showed only weak protection against oxidative damage as compared to alpha-tocopherol. In the presence of high concentrations of melatonin (1000 mol/mol LDL) in the medium a clear protective effect was found during lag- and propagation phase, albeit weaker than after loading with alpha-tocopherol. It is concluded that melatonin is not incorporated into LDL in sufficient concentrations to prevent lipid peroxidation effectively. When melatonin is present in the incubation medium during oxidation, a partitioning equilibrium between aqueous and lipid phase is established. Only under these conditions can melatonin act as a chain breaking antioxidant. The concentrations required, however, are far beyond those found in human plasma. Therefore, the data in this study do not support a direct physiological relevance of melatonin as an antioxidant in lipid peroxidation processes.

An in vivo long-term perfusion system is presented, which is based on automated, computer-controlled high-frequency heparin (10 U/mL) flushing of the cannula inserted into the tail artery of freely moving rats. The short flushing intervals as achieved with this system guarantee stable conditions throughout an experiment of several days, and reduce blood clotting and the infiltration of fibroblasts. In addition, a specially developed software enables the periodical application of test substances into the blood stream under time-controlled conditions over long periods. The system can be used continuously to follow up blood parameters in single animals without significant elicitation of stress-sensitive plasma catecholamines for up to 12 days.

Objective: To examine the usefulness of determining extended serum cytokine profiles in patients with juvenile rheumatoid arthritis (JRA), for the purpose of improving differential diagnosis and monitoring disease activity., Methods: In a 2-year prospective study, serum levels of interleukin-1 beta (IL-1 beta), soluble IL-2 receptor (sIL-2R), IL-6, IL-8, tumor necrosis factor alpha (TNF alpha), and the p55 soluble TNF receptor (sTNFR) were repeatedly determined by enzyme-linked immunosorbent assay in 40 patients with JRA, 13 patients with postinfectious arthropathies, and 30 healthy controls. The data were compared with conventional parameters of inflammation, such as C-reactive protein (CRP), iron and hemoglobin levels, erythrocyte sedimentation rate (ESR), white blood cell (WBC) counts, and platelet counts. WBC subsets were analyzed by flow cytofluorometry., Results: At the first visit and at the peak of inflammatory activity according to CRP levels and/or ESR, serum levels of sIL-2R, IL-6, and sTNFR in JRA patients correlated significantly with conventional inflammation indicators, whereas IL-1 beta, IL-8, and TNF alpha did not. No changes in leukocyte subset distribution were noted. Among the different clinical subtypes of JRA, sIL-2R, IL-6, and sTNFR values at the time of the initial visit showed a pattern similar to CRP, whereby patients with systemic disease exhibited by far the highest values. TNF alpha and IL-1 beta were variably elevated in certain JRA subtypes. Patients with postinfectious arthropathies showed elevated levels of CRP, sIL-2R, TNF alpha, and sTNFR, which did not differ significantly from levels in the various JRA subtypes with the exception of systemic disease. Detailed analysis of types I and II pauciarticular JRA revealed that levels of CRP, IL-1 beta, and TNF alpha were elevated in patients with type I disease. While these parameters were invariably normal in patients with type II disease, sTNFR and sIL-2R were still found to be significantly elevated. Followup studies suggested that persistently high sTNFR values are a better indicator of JRA activity than are measurements of other cytokines or CRP., Conclusion: JRA is associated with significant and consistent changes in serum levels of inflammatory cytokines and soluble receptors. For the clinical monitoring of JRA, determination of levels of sTNFR, and to some extent sIL-2R, may be particularly useful, since these determinations yield information about subtype and/or activity of disease that is not available from conventional parameters of inflammation.

In-vitro evidence suggests that DNA methylation may be involved in the development of forbidden immune responses that can result in autoimmune disease. In the present study we examined in-vivo effects of 5-azacytidine (5-azaC), a substance that inhibits DNA methylation, on the immune system and the occurrence of a spontaneous autoimmune disease in the chicken model. We found that (1) treatment of young normal chickens with 1.0 mg/kg 5-azaC on 7 consecutive days caused a rapid degeneration of the central lymphoid organs thymus and bursa; (2) this regimen with 5-azaC apparently inhibited B cell maturation, as the frequency of cytoplasmic Ig+ plasma cells in the bone marrow was found to be significantly reduced, whereas the total number of bone marrow cells was unchanged; and (3) a chronic low-dose (0.5 and 1.0 mg/kg) application of 5-azaC through 6 weeks was found to significantly enhance the spontaneous autoimmune thyroiditis in newly hatched chickens of the Cornell C strain, as determined by anti-thyroglobulin autoantibody titres and histological analysis of thyroid gland infiltration. The possible implications of these data for the generation of pathogenic autoimmune responses are discussed.

The purpose of this study was to examine patterns of peripheral T-cell-activation antigen expression after polyclonal in vitro stimulation in early stages of lymphoproliferative diseases. With 18 patients afflicted with recently diagnosed, non-leukemic stages of B and T cell lymphoma cytofluorimetric analysis was performed with peripheral blood lymphocytes (PBL) after 72 h in culture with and without phytohemagglutinin, using antibodies against the differentiation antigens CD3, CD8, CD4, CD16, CD19, CDw14, and the activation antigens interleukin-2 receptor (IL-2R, CD25), HLA-DR (DR), CD56 and transferrin receptor (TR). Compared to healthy controls and patients with other diseases, a very significant reduction of large T cells bearing activation markers was found in all lymphoma cases. Furthermore, a pronounced inhibition in the expression of the activation markers IL-2R and TR, but not of DR, was detected on CD3+ cells in phytohemagglutinin-stimulated PBL of all lymphoma cells independently of DNA synthesis, as measured by [3H]thymidine uptake. Determination of the natural-killer-cell-(NK)-associated antigens CD16 and CD56, available for our studies in a CD16 + CD56 combination kit, revealed, after phytohemagglutinin stimulation, significantly increased expression values in 8 lymphoma patients so far investigated, as compared to 12 healthy controls. Thus, polyclonal activation combined with cytofluorimetric screening of activation antigens seems to give useful information on the functional defect(s) of PBL in an early state of lymphoma, and may therefore be of considerable diagnostic value. The observed pattern of T cell activation antigen expression after phytohemagglutinin stimulation may give further clues to the understanding of immune dysfunction(s) associated with lymphoma.

Thyroglobulin autoantibody (Tg-AAb) can be spontaneously produced in vitro with thyroid infiltrating lymphocytes (TIL) collected from Obese strain chickens 3.5 and 4 weeks old. Attempts to enhance Tg-AAb synthesis with two known polyclonal stimulators of immunoglobulin synthesis in chickens, Staphylococcus aureus Cowan strain 1 and dextran sulphate, failed to increase Tg-AAb production in vitro. Spleen cells and peripheral blood lymphocytes obtained from the same chickens as the TIL and older chickens known to produce moderate to high levels of Tg-AAb in vivo did not produce autoantibody either spontaneously or in the presence of polyclonal Ig stimulators with one exception. With this single, exceptional chicken we obtained a small amount of Tg-AAb produced in vitro with spleen cells. This suggests that in the OS chicken TIL, and to a much lesser extent, the spleen, contribute to the total Tg-AAb produced in this model of autoimmune thyroiditis.

The exocytotic exposure of antigens of chromaffin granule membranes was studied with chromaffin cells isolated from bovine adrenal medulla. Antigens on the cell surface were visualized by indirect membrane immunofluorescence employing antisera against glycoprotein III and dopamine beta-hydroxylase. With unstimulated cells, only weak immunofluorescence on the cell surface was observed, whereas stimulated cells (with carbachol or Ba2+) exhibited much stronger reactions. In all cases the staining appeared as dots and patches. To quantitatively prove these observations, we analyzed the immunostained cells using a fluorescence-activated cell sorter. After stimulation, the average fluorescence intensity of the cell population was enhanced. This increase correlated with the degree of catecholamine secretion. The fluorescence intensity of stimulated cells varied over a broad range indicating that individual cells reacted variably to the secretagogues. When stimulated cells were incubated at 37 degrees C for up to 45 min after stimulation, a decrease of membrane immunofluorescence approaching that of unstimulated control cells was observed. Apparently, the membranes of chromaffin granules, which had been incorporated into the plasma membrane, were retrieved by a specific and relatively fast process. This retrieval of the antigen from the cell surface was blocked by sodium azide, but not influenced by colchicine, cytochalasin B, and trifluoperazine. The quantitative methods established in this paper should prove useful for further study of the kinetics of the exo-endocytotic cycle in secretory tissues.

The fluorescence kinetics of fluorescein isothiocyanate (FITC)-antibody conjugates during short (0.01 sec) laser excitations were analyzed for possible information about the immunological specificity of binding to antigenic substrates in direct immunofluorescence assays with antigen-coated Sephadex beads. First results of these investigations suggest marked differences in the bleaching characteristics of specifically and nonspecifically bound FITC conjugates: FITC-anti-bovine serum albumin (BSA) and FITC-anti-rabbit immunoglobulin (Ig) were found to fade significantly more slowly when bound to the respective homologous antigen (anti-BSA to BSA, and anti-rabbit Ig to rabbit Ig) as compared to nonspecifically adherent anti-BSA to rabbit Ig, and anti-rabbit Ig to BSA. Possible implications of these data for discrimination of nonspecific staining in immunofluorescence are discussed.

The present study provides first evidence for the presence of a thymic cell growth factor (TCGF) in supernatants (20 hrs) of mitogen (Con A) stimulated chicken spleen cells. 2 out of 3 batches of supernatant prepared according to a procedure originally described for the mouse system (10) induced a vigorous proliferative response of Con A prestimulated chicken spleen cells without significant mitogenic effect on unstimulated lymphocytes. No crossreactivity of this chicken-TCGF was observed with prestimulated murine lymphocytes, nor did potent mouse-TCGF preparations exhibit any proliferative effect on chicken cells. The implications of these data for both phylogenetic as well as practical experimental aspects are discussed.

In contrast to systemic autoimmunity, spontaneous autoimmune thyroiditis of Obese strain (OS) chickens is associated with a marked T cell hyperreactivity in vitro, i.e. an increased proliferation and interleukin 2 (IL 2) secretion in response to Concanavalin A (ConA). In the present study we report an enhanced capacity of OS peripheral lymphoid cells (splenocytes and peripheral blood lymphocytes, PBL) to adsorb fluorescein isothiocyante (FITC) labelled ConA, but not phytohemagglutinin (PHA). However, the elevated ConA binding cannot be a prerequisite for in vitro ConA hyperreactivity as OS thymocytes are normal with respect to ConA binding but nonetheless exhibit elevated responses to this mitogen. Moreover, ConA binding does not correlate with the frequency of cells able to express IL 2 receptors upon short term ConA stimulation. The percentage of ConA activatable cells was found to be increased in OS- PBL as compared to normal control PBL, but was unaltered in OS splenocytes. This finding points to a further mechanism of T cell hyperreactivity in OS chicks in addition to the previously reported defects in nonspecific immunosuppression. Finally, enumeration of cells in the S phase revealed that enhanced proliferation of OS T lymphocytes was not restricted to the in vitro response to ConA and phytohemagglutinin (PHA) but also occurs in vivo.

Frozen sections of chicken lymphoid organs were examined for lymphocyte surface antigens by antisera to T and B lymphocytes (ATS;ABS), and for the presence of immunoglobulin (Ig) G Fc and complement receptors (FcR;CR) by hemadsorption with sheep erythrocytes (E) coated with chicken IgG(EA), and E coated with rabbit IgG and chicken complement (EAC). In the spleen FcR positive cells were confined to the periellipsoidal sheaths and the germinal centers. CR positive cells were found in the same spleen areas, as well as in the medulla of bursal follicles. These lymphoid areas reacted strongly with ABS, but they also stained with neutral alpha-naphthyl butyrate esterase, and phagocytosed carbon particles were found in the periellipsoidal sheaths. Furthermore, in vivo treatment with cyclophosphamide, which resulted in pronounced B-lymphocyte depletion, did not affect FcR activity, but reduced CR activity significantly. These data indicate that the FcR activity demonstrated in tissue sections is mainly confined to mononuclear phagocytes, while the CR positive cells are mainly B lymphocytes.

Bleaching of stained objects is a major problem in immunofluorescence. The prevention of fluorescence fading would allow longer observation times, photographic documentation, fluorometry, and pattern recognition. Fluorescein kinetics and fluorescence intensities (FI) of fluorescein isothiocyanate (FITC) conjugate-stained Sephadex beads were studied with previously described "antibleaching" reagents using an argon laser as the excitation light source. Eight antibleaching reagents were tested (sodium azide (NaN3), sodium iodide (NaI), polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), 1,4-di-azobicyclo-(2,2,2)-octane (DABCO), p-phenylenediamine (PPD), n-propylgallate, and sodium dithionite (Na2S2O4]. Sodium azide and sodium iodide were found to increase FI. This was likewise found with mercury arc illumination and hence they may prove useful for routine immunofluorescence tests. PPD was found to accumulate on the surface of the beads and to disturb immunofluorescence by autofluorescence. The value of any of the other reagents in immunofluorescence is questionable.

Excitation and emission properties of fluorescein derivatives were studied macrofluorometrically. Measurements were performed with solutions of various concentrations (0.07-100 microgram/ml) of free sodium fluorescein prepared from fluorescein diacetate (FDA), fluorescein isothiocyanate (FITC) and FITC bound to rabbit gamma-globulin. Both excitation and emission spectra as well as fluorescence intensities at constant excitation/emission wavelengths (496/515 nm) were recorded. The findings indicate that (1) FDA gives about twice the fluorescence intensity compared to equal concentrations of FITC. (2) The fluorescence properties of FITC upon excitation with blue light (lambda = 496 nm) are only slightly altered by the conjugation to rabbit gamma-globulin. (3) Considerable quenching due to conjugation could, however, be shown to occur upon UV excitation (lambda = 340 nm). (4) Fluorescence emission excited by visible blue light (496 nm) increases linearly to dye concentration in a range of 0.07-2.5 microgram/ml. Beginning at 5 microgram/ml (10-(5) M/1) all three compounds show a sharp decrease of fluorescence intensity with further increasing concentration. Practical aspects of these data for the immunofluorescence method are discussed.