Simple dipstick assay for semi-quantitative detection of neopterin in sera

Neopterin, a low-molecular-mass pteridine produced by macrophages, is closely associated with activation of the cellular immune system. Neopterin biosynthesis during inflammatory disease is primarily derived from interferon-activated monocytes/macrophages and neopterin concentrations may be significantly increased in a particular disease state compared to controls. A follow-up of serum neopterin concentrations during the course of an infectious disease could be useful for measuring the activity of the disease and the influence of treatment. We have developed a simple dipstick assay for the semi-quantitative detection of the neopterin concentration in the serum of patients during the course of an infectious disease. Assay performance was comparable to an ELISA, but there is no requirement for specialised equipment.     

Sequential divergence and the multiplicative origin of community diversity

Phenotypic and genetic variation in one species can influence the composition of interacting organisms within communities and across ecosystems. As a result, the divergence of one species may not be an isolated process, as the origin of one taxon could create new niche opportunities for other species to exploit, leading to the genesis of many new taxa in a process termed “sequential divergence.” Here, we test for such a multiplicative effect of sequential divergence in a community of host-specific parasitoid wasps, Diachasma alloeum, Utetes canaliculatus, and Diachasmimorpha mellea (Hymenoptera: Braconidae), that attack Rhagoletis pomonella fruit flies (Diptera: Tephritidae). Flies in the R. pomonella species complex radiated by sympatrically shifting and ecologically adapting to new host plants, the most recent example being the apple-infesting host race of R. pomonella formed via a host plant shift from hawthorn-infesting flies within the last 160 y. Using population genetics, field-based behavioral observations, host fruit odor discrimination assays, and analyses of life history timing, we show that the same host-related ecological selection pressures that differentially adapt and reproductively isolate Rhagoletis to their respective host plants (host-associated differences in the timing of adult eclosion, host fruit odor preference and avoidance behaviors, and mating site fidelity) cascade through the ecosystem and induce host-associated genetic divergence for each of the three members of the parasitoid community. Thus, divergent selection at lower trophic levels can potentially multiplicatively and rapidly amplify biodiversity at higher levels on an ecological time scale, which may sequentially contribute to the rich diversity of life.Population divergence is a fundamental evolutionary process contributing to the diversity of life (1). Studies of how new life forms originate typically focus on how barriers to gene flow evolve in specific lineages, resulting in their divergence into descendent daughter taxa. As a result, evolutionary biologists now have a good understanding of how variation within a population is transformed by selection into differences between taxa (1–3). What is less well understood is whether the divergence of one population has consequences that ripple through the trophic levels of an ecosystem and affect entire communities of interacting organisms. Studies in paleontology (4–6), community ecology (7, 8), systematics (8, 9), and ecosystem genetics (10, 11) suggest that evolutionary change in one lineage can influence entire communities of organisms. For example, when the genotype/phenotype of a “foundation” species influences the relative fitness of other species, evolutionary change(s) in this genotype/phenotype may affect organisms in adjacent trophic levels (10, 11). If these evolutionary changes are linked to ecological adaptation and reproductive isolation (RI), associated organisms may diverge in parallel, potentially creating entire coevolved communities distinct from one another (12–15). Therefore, population divergence may not always be an isolated process, as the differentiation of one taxon could beget the divergence of many others.Such “sequential” or “cascading” divergence events may be particularly relevant to understanding why some groups of organisms, like plants, the insects that feed on them, and the parasitoids that attack the insects, are more diverse and species-rich than other groups (8, 9, 12–15). Specifically, when phytophagous insects diversify by adapting to new host plants, they create a new habitat for their parasitoids to exploit (Fig. 1). If a parasitoid shifts to the new habitat, it can encounter the same divergent ecological selection pressures as its insect host, which could result in the parallel divergence of insect host and parasitoid (12–15) (Fig. 1A). Moreover, sequential divergence may have multiplicative effects in generating biodiversity, as the shift of an insect to a new plant may open a new niche opportunity for not just one but the entire community of parasitoids attacking the insect host (12, 13) (Fig. 1B). However, few convincing examples of sequential divergence exist (12–15), and in no study is there both genetic and ecological evidence for sequential divergence multiplicatively amplifying biodiversity.Open in a separate windowFig. 1.Three scenarios of codivergence in a host−parasitoid system. (A) A single sequential divergence event, (B) sequential divergence with multiplicative amplification of biodiversity, and (C) cospeciation in allopatry. In A, codivergence is driven by the cascade of divergent ecological selection pressures across trophic levels in sympatry. Here, a degree of divergent ecological adaptation must accompany the host shift such that parasitoids are not merely moving between geographically separated hosts. In B, the multiplicative effects of sequential divergence can be seen as several members of the parasitoid community diverge in parallel with their host. In C, codivergence (cospeciation) occurs after host plant, fly, and parasitoid populations become jointly geographically isolated (black bar), resulting in parallel allopatric speciation. Here, little differentiation need accompany the initial host shift of fly or parasitoid. Cospeciation is not necessarily driven by the creation and adaptation to new niches but by the concordant geographic and reproductive separation of hosts and parasitoids.Here, we test for the multiplicative effects of sequential divergence in the community of parasitoid wasps (Hymenoptera: Braconidae) that attack fruit flies in the Rhagoletis pomonella sibling species complex (Diptera: Tephritidae) (Fig. 2). Several features of the biology and biogeography of the Rhagoletis−parasitoid system make it ideal for investigating the multiplicative divergence hypothesis and allow us to directly test multiple criteria supporting sympatric host race formation (16) and sequential divergence (12, 13), summarized in 1–3, 17–19), the hypothesized initial stage of ecological speciation (16, 17). The short time frame and sympatric spatial context of R. pomonella’s shift to apple exclude passive codivergence or speciation (Fig. 1C) as an explanation for differentiation. Specifically, fly and wasp populations could not have diverged in concert, because they became jointly geographically separated in the past (Fig. 1C). Rather, if flies and wasps display concordant adaptations, it is likely due to the direct effects of divergent ecological selection resulting from host shifts cascading from host plants to flies to parasitoids (Fig. 1 A and B).Open in a separate windowFig. 2.The community of host-specific parasitoids that attack members of the (A) Rhagoletis pomonella sibling species complex: (B) D. alloeum, (C) D. mellea, and (D) U. canaliculatus that (E) emerge from the fly pupal case as adults following overwintering. (Scale bar, 1 mm.)

Table 1.

Summary of conditions (criteria) conducive to and supporting hypotheses of sympatric host race formation (modified from ref. 16) and sequential divergence (modified from refs. 12 and 13)

Immunoglobulin-bearing lymphoid cells in primary syphilis. Quantitative and elution studies

The delay in antibody production in response to infection with Treponema pallidum may be caused by a block in the differentiation of antigen-stimulated B (Bursa-dependent) lymphoid cells towards plasma cells. This hypothesis was tested by a study to detect clonal expansion of immunoglobulin-bearing B lymphoid cells by in-vitro immunofluorescence tests in patients with primary syphilis. In addition, antibodies eluted from circulating lymphoid cells were investigated for treponemal binding by the enzyme-linked immunosorbent assay, the T pallidum immobilisation test, and the immunoglobulin class-specific FTA-ABS test. Results indicated that the number of IgG-bearing lymphoid cells were increased in patients with primary syphilis. However, in only a few cases could antitreponemal antibodies be eluted from isolated lymphoid cells. For this reason, the original hypothesis was rejected.     

Antibodies eluted from lymphoid cell membrane. Occurrence in certain varieties of scleroderma.

By means of acid elution two antibodies could be removed successfully from the circulating lymphocytes of 11 patients with certain varieties of scleroderma. One was specifically directed against nuclear antigen(s) of endothelial cells (NEC) of the dermal blood vessels, and another against nuclear antigen(s) of epidermal basal cells (NBC) of the involved and uninvolved skin of the patients. In two cases of acroscleroderma, the eluates failed to react with either endothelial or basal cells of involved or uninvolved skin. In none of 20 healthy controls involved in this study could an antibody be eluted from the circulating lymphocytes. The aforementioned antibodies do not bind complement in vitro and do occur in the serum of four patients. Circulating antinuclear antibody (speckled type) was detectable in two cases of scleroderma.     

Neopterin: a review

Neopterin was discovered in bee larvae, in worker bees and in royal jelly. The compound was termed "neopterin" to denote that it might start a new (Greek, neo) epoch in pteridine research. Increased concentrations of neopterin were reported in patients with viral infections, suggesting that increased neopterin may originate from the immune response of patients to the infections. In vitro studies revealed that human monocytes/macrophages produce neopterin when stimulated by interferon-gamma. Neopterin can easily be detected in serum and urine. The most important clinical applications for the determination of neopterin are prognostic indicator of malignant diseases, follow-up control of chronic infections, monitoring of immune-stimulatory therapy, differential diagnosis of acute viral and bacterial infections, prognostic indicator in HIV infections and early indications of complications in allograft recipients. In recent years new physiological functions of neopterin have been discovered such as inducing or enhancing cytotoxicity, inducing apoptosis and the role of a chain breaking antioxidant. This review will focus on the immunological and physiological properties of neopterin.     

Immunopathological studies on alopecia areata

Summary Anti-endothelial cell antibodies could be removed from circulating lymphocytes by means of acid elution techniques in eight patients with different degrees of alopecia areata. These antibodies were specifically directed against the endothelial cells in the capillary network of the hair bulb, indicating the existence of an antigen, which is unique to these particular endothelial cells. These antibodies do not bind complement in vitro and are species-specific.Circulating ANA (speckled type) were only noticed in case with alopecia areata in spots. A significant decrease in circulating T cells was noticed in six of eight patients with a certain degree of alopecia.Based on a paper presented at the 1st International Congress of Hair Research, Hamburg, March 1979     

Antitreponemal IgE in early syphilis

Using a solid-phase radioimmunoassay technique, mean serum IgE concentrations were found to be raised in patients with early syphilis. Antitreponemal specificity of the IgE response was investigated by the fluorescent treponemal antibody absorption test using a fluorescein-isothiocyanate-labelled antiserum against the Fc-fragment of human IgE. Validity of this test procedure was assessed by blocking experiments. The results provide evidence of the antitreponemal specificity of the IgE response in syphilis and indicate a possible role for antitreponemal IgE in the pathogenesis of the Jarisch-Herxheimer reaction and in the immune-complex origin of some of the lesions of secondary syphilis.     

Immunoglobulin- and complement-bearing lymphocytes in allergic contact dermatitis and atopic dermatitis (eczema)

The distribution pattern of immunoglobulin- or complement-bearing lymphocytes in healthy controls and in patients with contact dermatitis and with atopic dermatitis has been investigated. A significant predominance of IgD- and C₃-bearing lymphocytes was found in ten patients with contact dermatitis. In atopic dermatitis patients the number of IgE- and to a lesser extent of C₃-bearing lymphocytes was increased. The results indicate induction of stimulation of two distinct classes of immuno-logically competent lymphoid cell lines, one in contact and the other in atopic dermatitis. The increased number of B-cells in dermatitis indicates a decreased T-cell population and suggests a deficiency or disturbance of the recirculation of this population.     

Reappraisal of in situ immunophenotypic analysis of psoriasis skin: interaction of activated HLA-DR+ immunocompetent cells and endothelial cells is a major feature of psoriatic lesions

Psoriasis is an inflammatory skin disease of unknown aetiology. Many observations indicate that T cells play an important role in the pathogenesis of the disease. Upregulation of MHC class-II molecules on immunocompetent cells, endothelial cells and keratinocytes on lesional psoriatic skin has been regarded as a hallmark of the disease. However, there is some controversy in the literature regarding the cell types expressing class-II molecules and there is limited information about the presence of immune cells other than T cells and antigen presenting cells in the cellular infiltrates of psoriatic skin. We therefore reinvestigated the subject using immunocytochemical single and multiple staining techniques. In agreement with earlier reports, our studies showed that the cellular infiltrates in lesional skin consist largely of HLA-DR⁺/IL-2R⁺ T cells, HLA-DR⁺/CD1a⁺ Langerhans cells, and HLA-DR⁺/CD68⁺ macrophages. We found increased HLA-DR expression mostly on immunocompetent cells and endothelial cells, but no prominent HLA-DR expression on keratinocytes in lesional psoriatic skin. Upregulation of HLA-DR on endothelial cells and in mononuclear infiltrates was also evident in the non-lesional skin of psoriatic patients as compared with normal controls. B cells and natural killer cells were also found in the cellular infiltrates in lesional psoriatic skin. In spite of the presence of a large amount of activated T cells in the epidermis, we found that HLA-DR expression on keratinocytes was not a major feature of psoriatic skin.     

Immunoglobulin-bearing lymphoid cells in primary syphilis. Quantitative and elution studies.

The delay in antibody production in response to infection with Treponema pallidum may be caused by a block in the differentiation of antigen-stimulated B (Bursa-dependent) lymphoid cells towards plasma cells. This hypothesis was tested by a study to detect clonal expansion of immunoglobulin-bearing B lymphoid cells by in-vitro immunofluorescence tests in patients with primary syphilis. In addition, antibodies eluted from circulating lymphoid cells were investigated for treponemal binding by the enzyme-linked immunosorbent assay, the T pallidum immobilisation test, and the immunoglobulin class-specific FTA-ABS test. Results indicated that the number of IgG-bearing lymphoid cells were increased in patients with primary syphilis. However, in only a few cases could antitreponemal antibodies be eluted from isolated lymphoid cells. For this reason, the original hypothesis was rejected.