Chemokines in onchocerciasis patients after a single dose of ivermectin.

Ivermectin treatment will effectively diminish microfilariae (Mf) of Onchocerca volvulus in the skin of patients, but therapy is associated with adverse host inflammatory responses. To investigate the association of proinflammatory chemokines with the intensity of infection and clinical adverse reactions, chemokine serum levels were measured in patients following ivermectin treatment (100 microg/kg, 150 microg/kg or 200 microg/kg) or placebo. The density of O. volvulus Mf per mg skin decreased by 85%, 97%, 97% and 90% at day 3, at month 3, month 6 and at 1 year post-ivermectin. The cutaneous T cell-attracting chemokine (CTACK/CCL27) was found highly elevated in onchocerciasis patients compared to infection-free European controls (P = 0.0004) and it did not change following ivermectin or placebo to 1 year post-therapy. The chemokine RANTES/CCL5 (regulated on activated and normally T cell-expressed) was similarly high in onchocerciasis patients and infection-free European controls; the RANTES/CCL5 levels did not change following treatment until 6 months post-therapy but were slightly elevated at 1 year post-therapy (P < 0.02). In contrast, the Th2-type chemoattractants, thymus and activation regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22), were activated at 3 days post-ivermectin (P < 0.0001) to return to pretreatment or lower levels thereafter. The Th1-type chemoattractants, macrophage inflammatory protein (MIP)-1alpha/CCL3 and MIP-1beta/CCL4 were low before ivermectin treatment, but following clearance of microfilariae of O. volvulus their levels increased from 6 months post-therapy onwards (for both at 12 months post-therapy, P < 0.0001). The adverse reaction scores (RS) in treated patients increased significantly on day 3 (P < 0.02) while it remained unchanged in those who received placebo (P = 0.22); RS interacted with the microfilarial density (P = 0.01), but not with the dose of ivermectin or with the serum levels of MIP-1alpha/CCL3, MIP-1beta/CCL4, TARC/CCL17, MDC/CCL22 and CTACK/CCL27. Our observations suggest that following ivermectin, macrophages as well as memory Th2-type lymphocytes and B cells, attracted and activated by MDC/CCL22, TARC/CCL17 and CTACK/CCL27, may contribute to dermal immune responses and O. volvulus Mf killing and clearance. The transient changes of TARC/CCL17 and MDC/CCL22 were not associated with clinical adverse responses, and the later rise of MIP-1alpha/CCL3 and MIP-1beta/CCL4 showed a reactivation of Type 1 immune responses associated with persistent low levels of O. volvulus microfilariae and an expiring O. volvulus infection.

Assembly and clustering of acetylcholine receptors containing GFP-tagged epsilon or gamma subunits: selective targeting to the neuromuscular junction in vivo.

Acetylcholine receptor (AChR) gamma and epsilon subunits were tagged by green fluorescent protein (GFP) to analyse assembly and targeting in live muscle fibers at the neuromuscular junction. N- or C-terminal fusion polypeptides showed no fluorescence upon transfection of HEK cells. When GFP was inserted into the cytoplasmic loop connecting putative transmembrane regions M3 and M4, the gamma/GFP and epsilon/GFP subunits were fluorescent and formed together with the alpha, beta, and delta subunits GFP-tagged AChR complexes that were integrated into the plasma membrane. As the AChR were also clustered by rapsyn, the results indicate that the cytoplasmatic domains of the gamma and epsilon subunits may not be required for assembly and rapsyn-dependent clustering. The gamma/GFP and epsilon/GFP subunit-containing receptors were expressed in X. laevis oocytes and have affinities for acetylcholine similar to that of the wild-type receptors. Direct gene transfer into single muscle fibers reveals that gamma/GFP or epsilon/GFP polypeptides are expressed at the site of injection and are transported within the endoplasmatic reticulum. When reaching subsynaptic regions, both gamma/GFP or epsilon/GFP subunits compete with endogenous epsilon subunits to assemble GFP-tagged receptors, which are selectively targeted to the postsynaptic membrane.

Mutagenesis reveals a role for ABP/GRIP binding to GluR2 in synaptic surface accumulation of the AMPA receptor.

We studied the role of PDZ proteins GRIP, ABP, and PICK1 in GluR2 AMPA receptor trafficking. An epitope-tagged MycGluR2 subunit, when expressed in hippocampal cultured neurons, was specifically targeted to the synaptic surface. With the mutant MycGluR2delta1-10, which lacks the PDZ binding site, the overall dendritic intracellular transport and the synaptic surface targeting were not affected. However, over time, Myc-GluR2delta1-10 accumulated at synapses significantly less than MycGluR2. Notably, a single residue substitution, S880A, which blocks binding to ABP/GRIP but not to PICK1, reduced synaptic accumulation to the same extent as the PDZ site truncation. We conclude that the association of GluR2 with ABP and/or GRIP but not PICK1 is essential for maintaining the synaptic surface accumulation of the receptor, possibly by limiting its endocytotic rate.

Different functions of fetal and adult AChR subtypes for the formation and maintenance of neuromuscular synapses revealed in epsilon-subunit-deficient mice.

Mice deficient in epsilon-subunits of the acetylcholine receptor (AChR) channel die prematurely due to severe AChR deficiency that leads to the progressive reduction in AChR density at the neuromuscular endplate [Witzemann, V., Schwarz, H., Koenen, M., Berberich, C., Villarroel, A., Wernig, A., Brenner, H.R. & Sakmann, B. (1996) Proc. Natl Acad. Sci. USA, 93, 13286-13291]. The mice may serve as a model for studying AChR-related myasthenic diseases. The postnatal development of the subsynaptic apparatus takes place in the absence of the adult type, epsilon-subunit-containing receptors which normally replace the fetal gamma-subunit-containing receptors. During later development the secondary folds of the postsynaptic membrane disappear concomitant with the decrease in AChR density, so that the flattened-out membrane with its remaining nicotinic receptors is in close proximity to the subsynaptic cytoplasmatic compartment and the subsynaptic myonuclei. The decrease in AChR concentration is correlated with a decrease of postsynaptic rapsyn, but has less effect on agrin, a neuronally released aggregating factor for AChRs. Thus, despite the presence of agrin at the synapse, AChR expression is not maintained at the level required to stabilize normal synaptic structure comprising secondary postsynaptic membrane folds. Collectively the results suggest that the postnatal switch from the global, activity-sensitive gamma-subunit gene transcription to the synapse-specific, activity-independent epsilon-subunit gene transcription is not required for the formation and differentiation of synapses but is essential for the maintenance of the highly organized structure of the neuromuscular endplate.

Similar effects of electroporational stress and treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate on vimentin expression in mouse plasmacytoma cells.

In mouse plasmacytoma cells (MPC-11), an activation of the normally repressed vimentin gene was observed as a response to transfectional stress. Effects of electroporation on vimentin gene expression were compared at the cellular and chromatin level to those caused by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). At the cellular level, similar changes in vimentin gene activity and cell-cycle distribution were observed by flow cytometry, whereas at the chromatin level similar changes in patterns of hypersensitive regions were detected by DNase I mapping. Additionally, a region located 700 bp upstream of the transcriptional start became hypersensitive to DNase I digestion upon electroporation and TPA treatment. This region overlaps two adjacent AP-1-like binding elements and generates specific DNA/AP-1 complexes in bandshift experiments. Therefore, the transcription factor AP-1 seems to play a central role in the activation of vimentin gene expression induced by these 2 different forms of stress.

Role of actin cortex in the subplasmalemmal transport of secretory granules in PC-12 cells.

In neuroendocrine PC-12 cells, evanescent-field fluorescence microscopy was used to track motions of green fluorescent protein (GFP)-labeled actin or GFP-labeled secretory granules in a thin layer of cytoplasm where cells adhered to glass. The layer contained abundant filamentous actin (F-actin) locally condensed into stress fibers. More than 90% of the granules imaged lay within the F-actin layer. One-third of the granules did not move detectably, while two-thirds moved randomly; the average diffusion coefficient was 23 x 10(-4) microm(2)/s. A small minority (<3%) moved rapidly and in a directed fashion over distances more than a micron. Staining of F-actin suggests that such movement occurred along actin bundles. The seemingly random movement of most other granules was not due to diffusion since it was diminished by the myosin inhibitor butanedione monoxime, and blocked by chelating intracellular Mg(2+) and replacing ATP with AMP-PNP. Mobility was blocked also when F-actin was stabilized with phalloidin, and was diminished when the actin cortex was degraded with latrunculin B. We conclude that the movement of granules requires metabolic energy, and that it is mediated as well as limited by the actin cortex. Opposing actions of the actin cortex on mobility may explain why its degradation has variable effects on secretion.

Functional testosterone receptors in plasma membranes of T cells.

T cells are considered to be unresponsive to testosterone due to the absence of androgen receptors (AR). Here, we demonstrate the testosterone responsiveness of murine splenic T cells in vitro as well as the presence of unconventional cell surface receptors for testosterone and classical intracellular AR. Binding sites for testosterone on the surface of both CD4(+) and CD8(+) subsets of T cells are directly revealed with the impeded ligand testosterone-BSA-FITC by confocal laser scanning microscopy (CLSM) and flow cytometry, respectively. Binding of the plasma membrane impermeable testosterone-BSA conjugate induces a rapid rise (<5 s) in [Ca2+]i of Fura-2-loaded T cells. This rise reflects influx of extracellular Ca2+ through non-voltage-gated and Ni2+-blockable Ca2+ channels of the plasma membrane. The testosterone-BSA-induced Ca2+ import is not affected by cyproterone, a blocker of the AR. In addition, AR are not detectable on the surface of intact T cells when using anti-AR antibodies directed against the amino and carboxy terminus of the AR, although T cells contain AR, as revealed by reverse transcription-polymerase chain reactions and Western blotting. AR can be visualized with the anti-AR antibodies in the cytoplasm of permeabilized T cells by using CLSM, though AR are not detectable in cytosol fractions when using the charcoal binding assay with 3H-R1881 as ligand. Cytoplasmic AR do not translocate to the nucleus of T cells in the presence of testosterone, in contrast to cytoplasmic AR in human cancer LNCaP cells. These findings suggest that the classical AR present in splenic T cells are not active in the genomic pathway. By contrast, the cell surface receptors for testosterone are in a functionally active state, enabling T cells a nongenomic response to testosterone.

4Pi-confocal imaging in fixed biological specimens.

By combining the wavefronts produced by two high-aperture lenses, two-photon 4Pi-confocal microscopy allows three-dimensional imaging of transparent biological specimens with axial resolution in the 100-140-nm range. We reveal the imaging properties of a two-photon 4Pi-confocal microscope as applied to a fixed cell. We demonstrate that a fast, linear point deconvolution suffices to achieve axially superresolved 3D images in the cytoskeleton. Furthermore, we describe stringent algorithms for alignment and control of the two lenses. We also show how to compensate for the effects of a potential refractive index mismatch of the mounting medium with respect to the immersion system.

Estradiol binding to cell surface raises cytosolic free calcium in T cells.

The Fura-2 method is used to examine a possible action of 17beta-estradiol (E2) on [Ca2+]i of splenic T cells isolated from female C57BL/10 mice. E2 concentrations between 10 fM and 10 nM induce a rapid and dose-dependent increase in [Ca2+]i due to Ca2+ influx and release of Ca2+ from intracellular stores. Ca2+ influx is mediated by Ca2+ channels which are completely blockable by Ni2+ and partly by nifedipine. The antiestrogen tamoxifen does not inhibit the E2-induced rise in [Ca2+]i. Ca2+ influx and Ca2+ release from intracellular stores is also inducible by plasma membrane impermeable E2 conjugated to BSA. E2-BSA-FITC binds to the surface of T cells of both the CD4+ and CD8+ subset. Our data suggest a novel E2-signalling pathway in T cells which is not mediated through the classical nuclear estrogen receptor response but rather through putative plasma membrane receptors for E2.

TPA induces apoptosis in MPC-11 mouse plasmacytoma cells grown in serum-free medium.

Apoptotic-like events could be rapidly induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in cells of the mouse plasmacytoma cell line MPC-11 grown in serum-free medium. Indicators for apoptosis were morphological changes visualized by light and electron microscopy, such as chromatin condensation and the formation of cellular buds and fragments, as well as biochemical indices like the appearance of the so-called 'DNA ladder'. Additionally, in these cells which are usually devoid of significant amounts of cytoplasmic intermediate filament (cIF) proteins, synthesis and accumulation of the cIF protein vimentin was rapidly induced by TPA treatment and almost all cells became vimentin-positive. Later on, substantial amounts of vimentin and lamin B degradation products appeared, and an increasing fraction of cells displayed low or even undetectable quantities of intact vimentin. This subpopulation was characterized via microscopy to be in the late stages of apoptosis. We suggest that in MPC-11 cells undergoing apoptosis in response to TPA treatment vimentin as well as lamin B are degraded, leading to a rearrangement and eventual loss of their respective filament networks.

Schistosomiasis in women: manifestations in the upper reproductive tract.

Female genital schistosomiasis (FGS) is a neglected disease entity which may give rise to considerable suffering among women of child-bearing age in areas where schistosomiasis (especially due to Schistosoma haematobium) is prevalent. The close relation between the vessels in genital organs and the urinary bladder enables the parasite to easily change location to virtually any organs in the female pelvic area. Symptoms concur with the anatomical location of worm pairs and their ova. Lesions of the lower female genital tract can easily be investigated by cytology, histology or direct demonstration of eggs in scrapings or biopsies whereas schistosomiasis of the upper genital tract is clinically indecipherable and less accessible for examination. In the literature there are references to FGS as a cause of infertility, complications of pregnancy, menstrual disorders, problems related to sexual intercourse, diagnostic similarities to STDs and cancer, unspecified complaints related to blood loss, chronic abdominal pain, social segregation and related psychological problems. The diagnosis of female upper genital schistosomiasis is difficult and the authors point out possible diagnostic procedures which might be helpful for further understanding of this complex entity.

Female genital schistosomiasis due to Schistosoma haematobium. Clinical and parasitological findings in women in rural Malawi.

A total of 51 women with urinary schistosomiasis haematobium were examined in order to identify diagnostic indicators for female genital schistosomiasis (FGS). Patients were selected at random from the outpatient department of the Mangochi District Hospital, Malawi. The medical histories were recorded according to a pre-designed questionnaire and the women were subjected to a thorough gynaecological examination including colposcopy and photographic documentation of lesions. Microscopy of genital biopsies revealed that 33 of the 51 women had S. haematobium ova in cervix, vagina and/or vulva in addition to the presence of ova in urine. The most sensitive diagnostic procedure was beside microscopic examination of a wet cervix biopsy crushed between two glass slides, which revealed 25 of the 33 genital infections. There was a significant correlation between the size of genital lesions and the number of ova counted per mm2 of crushed tissue. Women with FGS had significantly more tumours in the vulva than women with schistosomiasis limited to the urinary tract. Most of the observed genital pathology could easily be identified by the naked eye, but colposcopic examination yielded valuable additional information like the demonstration of neovascularisation around cervical sandy patches. Few of the symptoms previously regarded as indicators for FGS could be linked to the presence of schistosome ova in genital tissue. Husbands of infertile women with FGS had children with other women significantly more often than husbands of women who only had urinary schistosomiasis. This, together with the finding that the majority of the divorced women had FGS, indicates that the manifestation of this disease may have implications for the marital and sexual life of the affected women.

Diagnosis of female genital schistosomiasis by indirect disease markers: determination of eosinophil cationic protein, neopterin and IgA in vaginal fluid and swab eluates.

Based on assumptions about the pathophysiology of egg-related lesions in the lower reproductive tract, putative indirect disease markers were investigated in vaginal fluids from 54 Malawi adolescent girls and women infected with S. haematobium. These women received a careful gynecological examination during which biopsies were taken from the cervix, and, if present, also from suspicious lesions in the vagina and the vulva. If the biopsies, either in wet crushed preparations or in histological sections, contained eggs the patients were considered to have female genital schistosomiasis (FGS; n = 33). The remainder (n = 21) were classified as having urinary schistosomiasis only. Eosinophil cationic protein (ECP), a cytotoxic granule protein of eosinophils, neopterin, a second messenger molecule generated during the activation of macrophages, and IgA as an indicator of local B-cell activation were quantitatively determined in vaginal fluid. To clarify the origin of ECP, this protein was also looked for in histological sections by an immunohistochemical method. In order to explore whether such disease markers can be detected after absorption to a tampon-like material, ECP and IgA were also assessed after elution from a non-porous, polypropylene fibre web impregnated with vaginal fluid. The concentration of ECP in vaginal fluid and the degree of immunohistochemical staining in histological sections were significantly higher in patients with FGS than in women with urinary schistosomiasis only. The amount of ECP detected in histological sections correlated to the number of eggs/mm2 of compressed genital tissue (rho = 0.36, P = 0.02), and the concentration of ECP in vaginal fluid correlated to the concentration of neopterin as well as to that of IgA (rho = 0.52, P = 0.004 and rho = 0.37, P = 0.02, respectively). Median neopterin concentration in vaginal fluid was also higher in the FGS group, but the difference was not statistically significant. ECP could also be detected in eluates from impregnated fibre webs, but the concentration was approximately one power of 10 less than in the original vaginal fluid. These results demonstrate that indicators of immunological mechanisms related to the egg-granuloma might be useful as indirect disease markers for women with FGS if assessed in vaginal washings or swab eluates.

Female genital schistosomiasis (FGS): relationship between gynecological and histopathological findings.

Schistosomiasis of the lower female reproductive tract manifests itself in a broad spectrum of clinical features. However, clinical and histopathological findings have never been studied in a synoptic manner. Based on the assumption that any type of pathology present in the female reproductive tract is the expression of a complex pathophysiological reaction towards eggs sequestered in the genital tissues, we decided to analyze colposcopic and histopathological findings in a comprehensive manner. Thirty-three women in Malawi with urinary and genital schistosomiasis were examined parasitologically and gynecologically. A thorough colposcopic examination with photodocumentation was performed and biopsies were taken from the cervix, the vagina and/or the vulva for histological sectioning and immunohistochemistry. The predominant colposcopic findings were sandy patches on the cervical surface similar to those seen in the bladder and polypous/papillomatous tumors with irregular surface on the vaginal wall and in the vulvar area. The histopathological sections of sandy-patch-like lesions demonstrated only a small cellular reaction around S. haematobium eggs in various stages of disintegration. In contrast, in the case of polyps the histology revealed a more pronounced immunological reaction characterized by a heavy cellular infiltrate. One case of invasive squamous cell carcinoma of the cervix was diagnosed. We conclude that colposcopy is a useful tool in the detection of FGS related pathology in the lower female reproductive tract and that the synoptic assessment of surface and of corresponding histological sections helped to understand the pathophysiology of S. haematobium associated disease in genital tissue.

Urine reagent strips for diagnosis of schistosomiasis haematobium in women of fertile age.

Hematuria, proteinuria and leukocyturia were semiquantitatively assessed by reagent strips in single morning urine of women of fertile age visiting the outpatient department of the Mangochi district hospital, Malawi. This was part of a diagnostic approach to female genital schistosomiasis (FGS). In 51 women ova of Schistosoma haematobium were detected in urine by a filtration technique. In 33 of these women ova were also present in genital tissue as demonstrated by microscopic examination of biopsies. In 209 women no ova were found in the single urine filtered. There were significantly higher scores for hematuria, proteinuria and leukocyturia as well as of the combined reagent strip index (RSI) in egg-excreting than in egg-negative women. The sensitivity of a single hematuria, proteinuria and leukocyturia reading was 98, 84 and 73%, respectively. However, the respective specificity was only 24, 22 and 23%. The best prediction of urinary schistosomiasis was achieved by a +2 score for hematuria, of which the sensitivity was 94% and the specificity was 61%. The high false-positive rates can probably be explained by contamination of urine by vaginal secretion. Moreover, cases of schistosomiasis have probably been overlooked because only a single morning urine sample was examined. The total absence of hematuria, proteinuria and leukocyturia, however, may be used to rule out heavy infections in community surveys. There was no difference in reagent strip scores between women with genital and urinary schistosomiasis as compared with those with urinary tract lesions alone. Thus urine analysis reagent strip readings do not help to discriminate between S. haematobium infected women with and without FGS.

Reversibility of lower reproductive tract abnormalities in women with Schistosoma haematobium infection after treatment with praziquantel--an interim report.

Little is known whether and to what extent antiparasitic treatment cures female genital schistosomiasis (FGS). Using a standard protocol, of twenty-one women with FGS nine were re-examined at two to nine weeks after they had been treated with praziquantel at a single dose of 40 mg/kg. Symptoms related to pathology of the urinary tract and to a lesser extent of genital pathology subsided in most patients. Schistosoma haematobium ova were no longer detectable in urine of any of the patients post-treatment. Efficiency of chemotherapy against adult worms was confirmed by the disappearance of circulating anodic antigen (CAA) in serum. Sandy patches showed resolution in two of four cases after chemotherapy. Papillomata due to schistosomiasis alone improved, but persisted in mixed infection with human papilloma virus (HPV) or when HPV was the only underlying cause. In one patient ulcera could not be related with certainty to schistosomiasis at admission, but resolved after treatment with parziquantel. Leukoplakia (two cases) was not influenced by chemotherapy, or even increased during follow-up, regardless of whether ova had been detected or not. Although the follow-up period was rather short, time intervals were not standardized, and a relatively small number of patients was investigated, it could be shown that genital pathology due to sequestered S. haematobium ova is, at least partially, reversible already two to nine weeks after killing the adult worms by praziquantel. This is paralleled by a normalization of inflammatory immune responses detectable in histological sections and vaginal lavage.