Does gold concentration in the blood influence the result of patch testing to gold?

BACKGROUND: We have recently found a correlation between contact allergy to gold sodium thiosulphate (GSTS) and gold concentration in the blood (B-Au) in a stented population: the higher the B-Au, the stronger the patch-test reaction. OBJECTIVES: To further investigate the correlation between B-Au and patch-test reactivity to gold. METHODS: In this provocation control cross-over trial of 24 patients with dermatitis with a known contact allergy to gold, the patients were randomized into two groups where one was topically provoked to gold (15 mg GSTS) and one to the control. All patients were simultaneously patch tested with GSTS in 10 aqueous dilutions (1.1 mg GSTS). Patch-test readings were performed and blood was drawn. After 6 weeks, the experiment was repeated and the group that had previously been provoked with gold was now provoked with the control and vice versa. RESULTS: B-Au was higher after gold provocation whereas no treatment effect was discerned for minimal eliciting concentration (MEC) or summarized test score (STS). Instead, significant differences in period effect were observed implying higher B-Au and STS and lower MEC on test occasion II. The most likely explanation is the increased B-Au and /or booster effect from test occasion I. There was a correlation between B-Au and MEC: the higher the B-Au, the lower the MEC. CONCLUSIONS: The correlation between B-Au and MEC indicates that the B-Au is of importance for the skin reactivity to gold.

Prolactin suppresses malonyl-CoA concentration in human adipose tissue.

Prolactin is best known for its involvement in lactation, where it regulates mechanisms that supply nutrients for milk production. In individuals with pathological hyperprolactinemia, glucose and fat homeostasis have been reported to be negatively influenced. It is not previously known, however, whether prolactin regulates lipogenesis in human adipose tissue. The aim of this study was to investigate the effect of prolactin on lipogenesis in human adipose tissue in vitro. Prolactin decreased the concentration of malonyl-CoA, the product of the first committed step in lipogenesis, to 77+/-6% compared to control 100+/-5% (p=0.022) in cultured human adipose tissue. In addition, prolactin was found to decrease glucose transporter 4 ( GLUT4) mRNA expression, which may cause decreased glucose uptake. In conclusion, we propose that prolactin decreases lipogenesis in human adipose tissue as a consequence of suppressed malonyl-CoA concentration in parallel with decreased GLUT-4 expression. In the lactating woman, this regulation in adipose tissue may enhance the provision of nutrients for the infant instead of nutrients being stored in adipose tissue. In hyperprolactinemic individuals, a suppressed lipogenesis could contribute to an insulin resistant state with consequences for the health.

Diffusion-in-gel thin layer immunoassay (DIG-TIA): optimal conditions for quantitation of antibodies.

A new technique for the quantitation of antigen-antibody reactions, diffusion-in-gel thin layer immunoassay (DIG-TIA), has been developed. The principle of DIG-TIA is that antibodies are allowed to form a concentration gradient by radial diffusion in an agar gel poured on top of an antigen-coated plastic surface. After removal of the gel and visualisation by means of condensation of water vapour on the plastic surface, the antigen-antibody reactions appear as zones of increased wettability. The concentration of antigen used for coating, pH of the agar, incorporation of Tween 20 in the agar, size of diffusion basins, time of diffusion, and reinforcement by anti-immunoglobulin have been studied with regard to their influence on sensitivity and precision in the detection of antibodies with DIG-TIA. A photographic technique for permanent recording of wettability patterns is also described.

A simple spot technique for thin layer immunoassays (TIA) on plastic surfaces.

A simple and sensitive technique for visualization of antigen--antibody reactions is described. The property of many antigens to become adsorbed firmly on to a hydrophobic polystyrene surface while retaining their serological reactivity is taken advantage of. On a surface with adsorbed antigen the corresponding immune serum is applied spot-wise. The antigen--antibody reaction areas on the surface are characterized by a distinct hydrophilic condensation pattern when exposed to water vapour. The results obtained by the immunoassay technique described can be reproduced with great accuracy. The method is well suited for quantitative determination of a wide range of antigens as well as their corresponding antibodies. Antigen concentrations of 0.2--0.8 mg/l and antibody concentrations about 1 mg/l can be detected. By employing an antiimmunoglobulin serum subsequent to the primary antigen--antibody reaction, an increase in sensitivity can be obtained.

Spot immunoprecipitate assay in gel compared with immune reactions in solution and applied to antibody titration.

Precipitation profiles of spot immunoprecipitate assay (SIA) reactions in gel are shown to have characteristics analogous to classic precipitin curves representing such immune reactions in solution. Thus SIA profiles of precipitated human albumin (HSA) and anti-HSA as checkerboard analysis permit: (1) determination of optimal proportions of antibody and antigen, (2) titration of antiserum as to antigen-binding capacity and (3) estimation of the concentration of specific antibody in an antiserum. The validity of the method was confirmed by similar results already reported for precipitin analysis of anti-HSA in solution. The stained protein assay method for determination of total protein used for the calculations had a reliable (r = 0.9979) working range for quantification between 0.1 and 0.83 microgram protein in 3 microliters samples (greater than or equal to 0.03 g/l). Study of SIA profiles in the extreme antibody excess region confirmed the validity of SIA quantification. The high sensitivity of SIA as compared with radial immunodiffusion is related to high ratios (8-9) of antibody to antigen; the reliable detection limit for SIA is 10 mg/l in a 3 microliters sample. SIA is easy to perform with simple laboratory equipment, allows measurement of any precipitable antigen with very small amounts of reactants, and gives results within an hour.

A novel two colour ELISPOT assay. I. Simultaneous detection of distinct types of antibody-secreting cells.

A novel assay system has been developed which is based on the ELISPOT methodology and employs a combination of two immunoenzyme visualization systems yielding distinct colour products. This variation permits the simultaneous enumeration of two different types of cell secreting antigenically distinct products. Optimal conditions for the concurrent detection of human mononuclear cells secreting IgG or IgA antibodies are described.

Solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of IgG rheumatoid factor-secreting cells.

Although IgG rheumatoid factor may play an essential role in the pathogenesis of rheumatoid arthritis, there is no precise method for its specific detection at the cellular level. A modification of the recently developed enzyme-linked immunospot assay has been devised for enumeration of cells secreting IgG rheumatoid factor (IgG RF) and simultaneous quantitation of the IgG RF secreted. Specific, sensitive and simple, this new assay should provide a valuable tool for study of isotype-specific RF secretion by single cells.

False positive results in class-specific rheumatoid factor (RF) assays due to interaction between RF and Fc fragments of anti-immunoglobulin indicator reagents.

A recently developed, simple immunological method, diffusion-in-gel ELISA, has been adapted for class-specific determination of rheumatoid factor (RF). Evidence was obtained of an analytical error due to interaction between RF and antigenic determinants on the Fc part of the indicator immunoglobulin (Ig) molecules used. This was eliminated by replacing the usual indicator reagent, complete Ig molecules, by conjugated Fab or F(ab')2 fragments. The findings imply that unless the RF-Fc interaction mentioned is avoided in the assaying technique, false positive results may be obtained for, e.g. IgG RF and IgA RF in sera containing IgM RF.

Reverse ELISPOT assay for clonal analysis of cytokine production. I. Enumeration of gamma-interferon-secreting cells.

A reverse modification of ELISPOT assay using nitrocellulose membranes and epitope-specific monoclonal antibodies is described for the detection of single lymphokine-secreting cells. As a model, the production of gamma-interferon by mitogen stimulated human peripheral blood lymphocytes has been examined. The assay can also be modified to permit microscopic examination of spot-forming cells.

Reverse enzyme-linked immunospot assay (RELISPOT) for the detection of cells secreting immunoreactive substances.

A reverse modification of the recently described enzyme-linked immunospot assay (ELISPOT), based on localized enzyme-substrate reactions in gel, is described for the enumeration of antigen-secreting cells using petri dishes coated with specific antibodies. As a model the detection of mouse and human immunoglobulin-secreting cells has been evaluated. Simple and sensitive, this new method, termed RELISPOT, can be adapted for the quantitation of secreted antigen thus providing additional information on the metabolic state of the population of cells tested.

A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells.

A solid-phase enzyme-linked immunosorbent assay (ELISPOT) is described for enumeration of cells secreting specific antibody. Spleen cells from immunized mice are incubated in antigen-coated polystyrene plates. After removal of the cells, bound antibodies are demonstrated by means of an immunoenzyme procedure in which enzyme-substrate reactions are performed in agarose. Dark-brown circular zones (spots), localized in areas of the dish where antibody production has occurred, are enumerated with the naked eye. Spectrophotometric estimation of enzyme-bound activity may be performed by substituting the gel for a liquid buffer, allowing accurate estimation of the total amount of secreted antibody. Versatile, sensitive and very easy to perform, this new assay provides a useful alternative to conventional plaque-forming cell assays.

Visceral leishmaniasis in Somalia. Significance of IgG subclasses and of IgE response.

We have determined the levels of IgG subclasses and IgE as well as specific antibodies of these isotypes in sera from 22 patients with clinical visceral leishmaniasis (VL) from Somalia. The results are compared with those obtained from 30 Somali and 23 Swedish controls. We found markedly increased concentrations of IgG1 in the VL sera, indicating that the pronounced increase in IgG in VL which is generally considered to be due to polyclonal B-cell activation is mainly restricted to this subclass. The IgG2 concentrations were significantly decreased. The IgG3 and IgG4 concentrations, on the other hand, did not differ between the two groups of Somali sera. The Somali control sera contained higher concentrations of IgG1 and IgG3, but significantly lower concentrations of IgG2 as compared to Swedish controls. The IgG4 values, on the other hand, were not different between the two groups of control sera. Anti-leishmania antibodies belonging to all IgG subclasses, were found in the patients' sera. There was no significant difference in total IgE between sera from VL patients and controls and specific IgE antibodies were only detected in a few patients. The Western blot assay (WB), revealed the presence of two bands corresponding to 74 kDa and 88 kDa in all patients' sera, indicating a possible diagnostic role for WB in this particular population.

The effect of immunomodulating treatment on cutaneous delayed-type hypersensitivity in MRL lpr/lpr mice.

An autoimmune disease in MRL lpr/lpr (MRL/l) mice is associated with a plethora of T-cell abnormalities, one of them being impaired delayed-type hypersensitivity (DTH). Three immunomodulating drugs, cyclophosphamide (Cy), cyclosporin A (CsA) and LS 2616, all with beneficial effects on autoimmune diseases, have been examined with regard to their potential effects on DTH in female MRL/l mice and in healthy control mice. All three drugs, given as a single dose at the time of sensitization, increased DTH reactivity of oxazolone in healthy control mice, while only two of them, Cy and LS 2616, significantly augmented the defective DTH response in MRL/l mice.

Application of thin layer immunoassay (TIA) for demonstration of antibodies against Entamoeba histolytica.

Thin layer immunoassay (TIA) was used to demonstrate antibodies against Entamoeba histolytica in sera from patients and blood donors. The TIA results agreed well with those obtained by the indirect hemagglutination (IHA) and immunodiffusion (ID) techniques. It is suggested that because of its technical simplicity and low cost TIA may be used alternatively or in addition to the IHA and ID techniques for screening patient sera for antibodies to E. histolytica. However, the new technique must first be evaluated on a goup of clinically well defined patients with different stages of amebiasis.

A comparison of thin layer immunoassay (TIA) and enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Trypanosoma cruzi.

The enzyme-linked immunosorbent assay (ELISA) was compared with a new solid phase method, thin layer immunoassay (TIA) for the serodiagnosis of Chagas's disease. 156 sera from Brazilian adult patients who were subject to investigation for chronic Chagas's disease, and 100 sera from healthy Swedish blood donors were analysed. There was a very good agreement between the methods with regard to positive and negative results and it is concluded that either method could be used for sero-diagnosis of Chagas's disease depending upon the choice and facilities of the particular laboratory.

Paper discs impregnated with capillary blood. A sampling technique for immunoassays by means of DIG-ELISA and DIG-TIA.

A modification of the diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA) and the diffusion-in-gel thin layer immunoassay (DIG-TIA) using paper discs impregnated with blood or serum as source of diffusion is described. Using a BSA-anti-BSA model system the suitability of this sampling and assaying technique for simple and accurate quantitation of antibodies has been demonstrated. Paper discs impregnated with blood or serum and subsequently dried could be stored at temperatures between +4 degrees C and +37 degrees C for at least six weeks without significant loss of antibody activity. Application of paper disc DIG-ELISA was exemplified by assaying anti-BSA antibodies in rabbits immunized with BSA as well as antibodies against both soluble egg antigen and adult worm extract in mice experimentally infected with Schistosoma mansoni cercariae. The simplicity and good reproducibility of this technique should make it an attractive alternative in serological screening of large population groups. Definite advantages of this technique are the possibilities to analyse quantitatively antibodies in capillary blood, rather than serum from venous blood samples, and to store sampled material for a considerable time even under such climatic conditions as often prevail in tropical countries.

Application of thin layer immunoassay (TIA) as a serodiagnostic tool in schistosomiasis. A preliminary report.

Thin layer immunoassay (TIA) is a recently developed simple serological technique which, in a preliminary study, has been applied to the serodiagnosis of schistosomiasis. 64 of a total of 69 sera from patients with schistosomiasis were positive in TIA when a Schistosoma mansoni worm antigen preparation was used as coating material. A trial of the diagnostic specificity of the assay was made by cross-testing sera from patients with different parasitic diseases, using TIA plates coated with extracts from the relevant parasites. All sera from patients with filariasis, fascioliasis and echinococcosis were positive in homologous TIA systems. In heterologous systems, cross-reactivity was noted for some sera in all groups except fascioliasis. The largest proportion of cross-reacting sera was registered in the echinococcosis group. It is suggested that, after further evaluation, the TIA technique might supplement or be used as an alternative to other serological tests already in use for the diagnosis of schistosomiasis. In favour of the TIA technique are its simplicity and its low cost.

Polypropylene fibre web, a new matrix for sampling blood for immunodiagnosis of schistosomiasis.

Blood sampling on filter paper is widely used for immunodiagnostic and epidemiological purposes. However, elution of conventional filter papers impregnated with sera containing Schistosoma mansoni circulating anodic antigen (CAA) recovered only a small fraction of the antigen, thereby reducing the sensitivity of the assay. Polypropylene-based non-woven fibre web is a new sampling material with a low density of fibres and with a small surface area of contact. When it was impregnated with serum containing CAA, approximately 90% of the antigen could be extracted. The yield of antibodies against S. mansoni from the new sampling material did not differ from that from conventional filter papers.

Visceral leishmaniasis in Somalia: prevalence of leishmanin-positive and seropositive inhabitants in an endemic area.

In an endemic area of Somalia both humoral and cell mediated immunity against Leishmania donovani was demonstrated in 246 inhabitants. In a study of 14 patients with active visceral leishmaniasis, we found that antibodies appear early in infection and that they are then demonstrable for a limited period only. Leishmanin positivity develops later and persists longer, but does not seem to be lifelong. The majority of the immunoreactive individuals were either sero- or leishmanin positive. This finding is in accord with the result obtained in recent experimental studies indicating a regulatory effect exerted on humoral and cell mediated immunity by different T lymphocyte subsets.