Actinic keratosis and surrounding skin exhibit changes in corneocyte surface topography and decreased levels of filaggrin degradation products.

Actinic keratosis (AK) is a frequent premalignant skin lesion mainly caused by chronic sun exposure. AK lesions are often surrounded by invisible, subclinical alterations, called field of cancerization (FoC). Definition of FoC is of importance for therapy management; however, the criteria and non-invasive tools to characterize FoC are lacking. Atomic force microscopy (AFM) proved to be a suitable tool for detection of changes in the corneocyte surface topography in inflammatory skin diseases, which share similar clinical features with AK such as hyper- and parakeratosis. Therefore, in this study we applied AFM to investigate AK and surrounding skin obtained by non-invasive collection of the stratum corneum (SC) with adhesive tapes. Furthermore, we determined degradation products of structural protein filaggrin (natural moisturizing factor, NMF), which previously showed association with the changes in corneocyte surface topography. Ten patients with multiple AK on the face were recruited from the outpatient clinic. SC samples were collected from the AK lesion, skin sites adjacent to the AK, 5 cm from the AK and retroauricular area. Corneocyte surface topography was determined by AFM, and NMF by liquid chromatography. The AK lesion showed alterations of the corneocyte surface topography characterized by an increased number of nanosize protrusions, which gradually decreased with the distance from the lesion. NMF levels show an inverse pattern. Atomic force microscopy showed to be a suitable tool to detect changes in the corneocyte surface topography on the AK lesion and surrounding skin in a non-invasive manner.

Impact of superior ophthalmic vein thrombosis: a case series and literature review.

Purpose: To present nine new cases of superior ophthalmic vein thrombosis (SOVT) and compare these with the literature, and to assess the impact of SOVT for the clinician. Methods: Using the data bases of the Department of Ophthalmology of the AMC, we searched for patients with radiologically evidenced SOVT between January 2006 and December 2014. In addition, a PubMed search, using the mesh term 'superior ophthalmic vein thrombosis', was done. Results: We found nine patients with SOVT. In three patients, SOVT was related to dural arteriovenous fistulae. In one patient, it was caused by the acute reversal of warfarin by vitamin K. In two patients, an infectious cause was found. In three patients, the cause of SOVT was not found despite screening for coagulation and other disorders. All patients presented with eyelid swelling, proptosis, and/or motility impairment. We found complete recovery in four patients. Three patients had mild sequelae and two patients had severe visual impairment. In the literature, we found 60 cases reporting on SOVT with various aetiologies. Clinical presentation, treatment modalities, and outcomes were comparable to our findings. Conclusion: Our case series and literature review show that SOVT can occur simultaneously with cavernous sinus thrombosis (CST) but can also be a separate entity. Clinical presentation can mimic orbital cellulitis (OC) or CST and when no signs of OC can be found, an alternative cause for SOVT should be sought. When timely and adequate treatment is conducted, the prognosis is predominantly favourable.

Antigen-coupled beads adherent to slides: a simplified method for immunological studies.

Small amounts of antigen-coupled beads adherent to object slides provide a simple, quick, economical and sensitive immunohistochemical means of detecting antibodies in serum by both immunofluorescence and immunohistoperoxidase procedures. Sensitivity increases with decreasing quantities of antigen-coupled beads, as was demonstrated in the fluorescence procedure.

Application of the FITC-anti-FITC-gold system to ultrastructural localization of antigens.

We report the application of a fluorescein isothiocyanate (FITC)-anti-FITC method to localize antigens at the ultrastructural level. In the systems studied, the anti-FITC-based detection method displays high specificity and sensitivity. These observations, combined with ease of production and with availability of FITC-protein conjugates, suggest that the FITC-anti-FITC method is a good alternative to presently used methods and is widely applicable to immunochemical and immunocytochemical procedures. The same preparation and protocol can be used for light and electron microscopic studies, thereby reducing possible artifacts introduced if different procedures are used. In the present study, two systems were used to test the method. One system used an FITC-labeled monoclonal antibody (MAb) to schistosome circulating cathodic antigen. In this system, the label was detected in the gut of adult Schistosoma mansoni by an anti-FITC MAb conjugated to 10-nm gold particles. The second system used human IgM antibodies pooled from patients infected with Schistosoma mansoni. In this system detection was accomplished using an anti-human IgM-FITC conjugate followed by the anti-FITC-Au antibody conjugate.

Immunologic characterization of two monoclonal antibodies reactive with repetitive carbohydrate epitopes of circulating Schistosoma mansoni egg antigen.

A panel of 60 monoclonal antibodies (MAbs) reactive with repetitive epitopes of species-specific Schistosoma mansoni soluble egg antigen (SEA) was tested for performance in detecting circulating egg antigens. Two MAbs, 114-5B1-A and 114-4D12-A, which were highly reactive with two different repetitive carbohydrate epitopes of soluble egg antigen, were found to detect circulating egg antigen in the sera of S. mansoni-infected mice. The two MAbs also showed strong reactivity with two high M(r) cercarial antigens present on the cercarial and schistosomular surface, while in the adult worms, antigens in the parenchyma were recognized. In two sandwich enzyme-linked immunosorbent assays (ELISA-5B1 and ELISA-4D12), each MAb was used as capture antibody and as conjugate, which resulted in assays with a lower detection level (0.2-0.4 ng) of the trichloroacetic acid-soluble fraction of soluble egg antigen (SEA-TCA)/ml. The antigen component(s) detected by ELISA-5B1 and ELISA-4D12 were 10,000 and 40,000 times more concentrated in the egg antigen than in the adult worm antigen, respectively. With both assays, in serum of heavily S. mansoni-infected mice, antigen became detectable from eight weeks postinfection (PI) onwards, with a striking increase at nine weeks PI.

Quantitative determination of circulating antigens in human schistosomiasis mansoni using an indirect hemagglutination assay.

In serum and urine specimens collected from a group of Schistosoma mansoni infected individuals from Makundju, Zaire, the schistosome circulating anodic antigen (CAA) and the circulating cathodic antigen (CCA) were quantitatively determined using an indirect hemagglutination reaction with sheep erythrocytes sensitized with mouse IgM monoclonal antibodies directed against these circulating antigens. Levels of CAA in serum (up to 5 ng/ml) and CCA in serum and urine (up to 50 ng/ml) were strongly correlated with egg excretion and with each other. No correlation was found between egg excretion and antibody levels against the circulating antigens. Antigen was detectable only in patients excreting greater than 500 eggs per gram of feces.

Applicability of different antigen preparations in the enzyme-linked immunosorbent assay for schistosomiasis mansoni.

The applicability of seven different antigen preparations for the detection of antibodies against Schistosoma mansoni was tested in the enzyme-linked immunosorbent assay. For this purpose, sera from children and adults from Surinam infected with S. mansoni were screened for the presence of specific antibodies against the various antigens. With all antigens, generally better results were obtained with the sera from children than with those from adults. The best results were obtained when the trichloroacetic acid-soluble fraction of adult worm antigen (containing the proteoglycan circulating anodic antigen) was used.

Detection of the schistosome circulating cathodic antigen by enzyme immunoassay using biotinylated monoclonal antibodies.

We have developed an enzyme immunoassay (ELISA) for the quantification of the schistosome circulating cathodic antigen (CCA), a glycoprotein associated with the syncitium lining the gut of the parasite. A mouse monoclonal antibody of IgG3 isotype was used as coating (antigen-capture) antibody, while a biotinylated mouse monoclonal IgM was used as second (antigen-detecting) antibody. Streptavidin-alkaline phosphatase was used as enzyme label. The lower detection limit of the assay was 1.0 ng of the trichloroacetic acid soluble fraction of adult worm antigen (AWA-TCA) per ml, which corresponds to approximately 0.2 ng CCA per ml. The ELISA showed a linear range from 1.0 to 62.5 ng AWA-TCA per ml. Serum and urine samples of 16 individuals infected with Schistosoma mansoni (egg counts ranging from 5 to 4820 eggs per gram of faeces) were tested in the assay. Antigen titres ranged from less than 4-8192. This assay represents a considerable advantage in diagnosis of Schistosoma infections as it allows the detection and quantification of CCA in serum and urine in even lightly infected individuals.

Evaluation of an ELISA for combined measurement of CAA and CCA in schistosomiasis mansoni.

An enzyme-linked immunosorbent assay was developed for combined measurement of schistosome circulating anodic antigen (CAA) and circulating cathodic antigen (CCA). Monoclonal antibodies against CAA and CCA were used as coating and as fluorescein-labeled detecting antibodies in a FITC-anti-FITC system. The lower detection limit of the assay was 1.1 ng antigen (AWA-TCA)/ml. Serum samples of Schistosoma mansoni infected individuals from Zaire (n = 60) and Burundi (n = 60) were tested in this assay and in single-antigen ELISAs. Sensitivities of assaying for CAA, CCA, combined CAA + CCA, and of parallel testing for CAA and for CCA were calculated from titres and antigen concentrations. With serum samples from the heavily infected individuals (Zaire), all assays had a sensitivity of 97% or higher. In contrast, with serum samples from individuals from Burundi (low to moderate infections) it was shown that combined testing resulted in a slightly lower sensitivity than testing for individual antigens. By parallel testing for CAA and CCA, the sensitivity could be increased considerably (to 95%), however.

Mapping fucosylated epitopes on glycoproteins and glycolipids of Schistosoma mansoni cercariae, adult worms and eggs.

The developmental expression of the antigenic fucosylated glycan motifs Fucalpha1-3GalNAcbeta1-4GlcNAc (F-LDN), Fucalpha1-3GalNAcbeta1-4(Fucalpha1-3)GlcNAc (F-LDN-F), GalNAcbeta1-4(Fucalpha1-3)GlcNAc (LDN-F), Galbeta1-4(Fucalpha1-3)GlcNAc (Lewis X), and GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc (LDN-DF) in Schistosoma mansoni cercariae, adult worms and eggs, was surveyed using previously defined anti-carbohydrate monoclonal antibodies (mAbs). Lewis X was found both on glycolipids and glycoproteins, yet with completely different expression patterns during the life-cycle: on glycolipids, Lewis X was mainly found in the cercarial stage, while protein-conjugated Lewis X was mainly present in the egg stage. Also protein-conjugated LDN-F and LDN-DF were most highly expressed in the egg-stage. On glycolipids LDN-DF was found in all three examined stages, whereas LDN-F containing glycolipids were restricted to adult worms and eggs. The motifs F-LDN and F-LDN-F were found both on glycoproteins and glycolipids of the cercarial and egg stage, while in the adult stage, they appeared to occur predominantly on glycolipids. Immunofluorescence assays (IFA) showed that these F-LDN and F-LDN-F containing glycolipids were localized in a yet undefined duct or excretory system of adult worms. Murine infection serum showed major reactivity with this adult worm duct-system, which could be fully inhibited by pre-incubation with keyhole limpet haemocyanin (KLH). Clearly, the use of defined mAbs provides a quick and convenient way to map expression profiles of carbohydrate epitopes.

A comparison of the IFA and the ELISA for the demonstration of antibodies against schistosome gut-associated polysaccharide antigens in schistosomiasis.

The applicability of two immunodiagnostic techniques was studied for the detection of antibodies against schistosome gut-associated polysaccharide antigens in human schistosomiasis mansoni: the immunofluorescent antibody reaction (IFA) using Rossman's fixed paraffin sections of adult worms and the enzyme-linked immunosorbent assay (ELISA) with a trichloroacetic acid soluble fraction of total adult worm antigens (AWA-TCA). With the IFA, gut-associated polysaccharide antigens could be demonstrated with an anti-IgM conjugate in a high percentage of the sera tested, although false-negative reactions were occasionally recorded. The use of an anti-IgG conjugate resulted in the demonstration of antibodies against additional antigens in the parenchyma of the worm and on the tegument. Specific IgM antibodies were present in higher concentrations in the sera from children than in those from adults. Using AWA-TCA as the antigen preparation in the ELISA, only antibodies against the circulating anodic antigen (CAA) could be demonstrated. Pretreatment of the ELISA-plates with poly-L-lysine to couple AWA-TCA was not necessary. The ELISA was successfully applied with anti-Ig, anti IgG, and anti-IgM conjugates. With anti-Ig conjugate the test was very sensitive and gave less false-negative reactions than the IFA. There was a significant difference between Ig, IgG, and IgM titres of children and adults. The use of an immunogalactosidase assay with a fluorogenic substrate in the ELISA, resulted in a test which was able to detect antibodies at ten times higher dilutions than with the immunoperoxidase assay.

Immunodiagnosis of recently acquired Schistosoma mansoni infection. A comparison of various immunological techniques.

A group of Dutch tourists, who became infected with Schistosoma mansoni in Ethiopia, was investigated in a serological follow-up study, during 8-50 weeks after infection. The following immunodiagnostic tests were applied: (1) the immunofluorescent antibody (IFA) test, both on frozen sections of adult worms, and in a modification for the detection of antibodies against gut-associated polysaccharide antigens; (2) the enzyme-linked immunosorbent assay (ELISA) with as antigens: adult worm antigens (AWA), cercarial antigens (CA), soluble egg antigens (SEA), and the purified antigens CAA and MSA1; (3) the defined antigen substrate spheres system with AWA as antigen in an immunofluorescence and immunoperoxidase modification; (4) the indirect haemagglutination reaction with AWA; and (5) the immunoelectrophoresis with AWA and antigens of the intermediate host. With these techniques it could be shown that in all persons which had been in contact with S. mansoni infected water, also in those not excreting schistosome eggs or not showing clinical symptoms of infection, specific anti-schistosome antibodies were present. No false-negative reactions were found with the ELISA with cercarial antigens, MSA1, or AWA-TCA, with the IFA detecting gut-associated polysaccharide antigens and with the immunoelectrophoresis. The highest titres were observed with the two techniques (IFA and ELISA) detecting antibodies against the gut-associated polysaccharide antigen CAA.

Immunodiagnosis of human schistosomiasis using different immunoprecipitation techniques.

One hundred sera of individuals infected with Schistosoma mansoni and/or S. haematobium were examined for the presence of specific anti Schistosoma antibodies by means of different immunoprecipitation techniques: immunoelectrophoresis, immunodiffusion, immunoelectroosmophoresis (on two different supports), and electroimmunodiffusion. The immunoelectroosmophoresis proved to be superior to the other immunoprecipitation techniques, its main advantages being sensitivity, rapidity, and economic use of reagents. Precipitins against the antigen of the intermediate host, Biomphalaria glabrata, were demonstrated in 66% of the sera.

Schistosoma mansoni: analysis of monoclonal antibodies reactive with gut-associated antigens.

The analysis of a series of monoclonal antibodies (mAbs) developed in our laboratory against gut-associated antigens of Schistosoma mansoni is described. It was found that mAbs that recognized epitopes of antigens in the gut and on the eggshell were mainly of the IgM isotype; these epitopes are likely to be carbohydrate in composition. Of a number of mAbs that were reactive with antigens important to the human humoral immune response, 75% appeared to be reactive with the circulating cathodic antigen.

Schistosoma: analysis of monoclonal antibodies reactive with the circulating antigens CAA and CCA.

Using spleen cells of mice infected or immunized respectively with cercariae or antigen preparations of Schistosoma mansoni, S. haematobium or S. japonicum monoclonal antibodies (mAbs) were produced against the schistosome gut-associated antigens CAA (circulating anodic antigen) and CCA (circulating cathodic antigen). Fusions nearly exclusively produced either anti-CAA (n = 25) or anti-CCA mAbs (n = 55) with a strong isotype restriction (IgM, IgG1 and IgG3) against both antigens, the majority of anti-CAA mAbs being IgG1 and the majority of anti-CCA mAbs being IgM. The mAbs, which on the basis of their selection were reactive with multiple carbohydrate epitopes of CAA or CCA, were applied in different immunological techniques including immunofluorescence, a dot immunobinding assay and immunoelectrophoresis to study the epitope repertoire. Anti-CAA mAbs were found to be reactive with 5 different epitopes, none of which occurred as multiple epitopes on eggs. Anti-CCA mAbs, on the other hand, recognized at least 10 different epitopes, while 44% of anti-CCA mAbs recognized epitopes common to the adult worm and the egg. Both CAA- and CCA-epitopes were found to be developmentally expressed at the level of the tegument in cercariae, schistosomula and 5-day-old lung worms, but in the adult worm were primarily found in the gut. Thus, the production of panels of mAbs has not only resulted in the selection of reagents optimally performing in diagnostic immunoassays, but also allowed a more detailed study of the epitope repertoire of these important schistosome antigens.