Differential distribution and biochemical characteristics of hydrolases among developmental stages of Schistosoma mansoni may offer new anti-parasite targets.

Schistosoma mansoni enzymes play important roles in host-parasite interactions and are potential targets for immunological and/or pharmacological attack. The aim of this study was to comparatively assess the presence of hydrolytic activities (phosphatases, glycosidases, aminopeptidases) in soluble (SF) and membrane (MF) fractions from different S. mansoni developmental stages (schistosomula 0 and 3h, juveniles, and adult worms of 28 and 45days-old, respectively), by using simple enzyme-substrate microassays. Our results show and confirm the prominent presence of alkaline phosphatase (AlP) activity in the MF of all the above parasite stages, highlighting also the relevant presence of MF-associated α-mannosidase (α-MAN) activity in juveniles. A soluble AlP activity, together with ß-N-D-acetylglucosaminidase (ß-NAG), and α-MAN activities, was detected in SF of schistosomulum 0h. Soluble ß-NAG, α-MAN, acid phosphatase (AcP), leucin (LAP) and alanine (AAP) aminopeptidase activities were also seen in the SF of the other different developmental stages. This work shows different soluble and membrane-associated hydrolytic capacities in each S. mansoni developmental stage from schistosomula to adults that might be exploitable as potential new targets for immune and/or chemoprophylactic strategies.

Comparative evaluation of Schistosoma mansoni, Schistosoma intercalatum, and Schistosoma haematobium alkaline phosphatase antigenicity by the alkaline phosphatase immunoassay (APIA).

To know if alkaline phosphatase (AP) from schistosomes other than Schistosoma mansoni can be used as diagnostic marker for schistosomiasis in alkaline phosphatase immunocapture assay (APIA), we comparatively tested n-butanol extracts of adult worm membranes from a Venezuelan (JL) strain of S. mansoni (Ven/AWBE/Sm); a Cameroonian (EDEN) strain of Schistosoma intercalatum (Cam/AWBE/Si) and a Yemeni strain of Schistosoma haematobium (Yem/AWBE/Sh). APIA was evaluated with sera of patients from Venezuela, Senegal, and Gabon infected with S. mansoni, from Gabon infected with S. intercalatum or S. haematobium, from Chine infected with Schistosoma japonicum and from Cambodian patients infected with Schistosoma mekongi. Results indicate that 92.5% (37/40) of Venezuela sera, 75% (15/20) of Senegal sera, 39.5% (17/43) of S. haematobium sera, and 19.2% (5/26) S. intercalatum sera were APIA-positive with the Ven/AWBE/Sm preparation. APIA with the Cam/AWBE/Si preparation showed that 53.8% of S. intercalatum-positive sera had anti-AP antibodies, and 51.2% S. haematobium-positive sera cross-immunocapturing the S. intercalatum AP. APIA performed with Yem/AWBE/Sh showed that 55.8% S. haematobium sera were positive. Only two out of nine S. japonicum sera were APIA-positive with the Ven/AWBE/Sm and Cam/AWBE/Si, and no reaction was observed with Cambodian S. mekongi-positive sera. AP activity was shown to be present in all the schistosome species/strains studied. The use of APIA as a tool to explore the APs antigenicity and the presence of Schistosoma sp. infections through the detection of anti-Schistosoma sp. AP antibodies in a host, allowed us to demonstrate the antigenicity of APs of S. mansoni, S. intercalatum, and S. haematobium.

Fosfatasa alcalina como marcador bioquímico de la enfermedad periodontal / Alkaline phosphatase as a biochemical marker of periodontal disease

La fosfatasa alcalina (ALP) es una enzima relacionada con la enfermedad periodontal (EP). Se encuentra en los polimorfonucleares (PMN), osteoblastos, fibroblastos y diversas céluals del tejido conjuntivo. Juega un papel importante en el remodelado del tejido óseo y del ligamento periodontal. Los niveles de ALP son elevados en los sitios con pérdida de inserción, permitiendo el diangóstico de EP y la vigilancia del tratamiento periodontal

Fosfatasa alcalina como marcador bioquímico de la enfermedad periodontal / Alkaline phosphatase as a biochemical marker of periodontal disease

La fosfatasa alcalina (ALP) es una enzima relacionada con la enfermedad periodontal (EP). Se encuentra en los polimorfonucleares (PMN), osteoblastos, fibroblastos y diversas céluals del tejido conjuntivo. Juega un papel importante en el remodelado del tejido óseo y del ligamento periodontal. Los niveles de ALP son elevados en los sitios con pérdida de inserción, permitiendo el diangóstico de EP y la vigilancia del tratamiento periodontal(AU)

Antibody-based detection of alkaline phosphatase in lepidopteran insects (Lepidoptera: Noctuidae).

Alkaline phosphatase (ALP) of Helicoverpa armigera Hub. (Lepidoptera; Noctuidae) (GenBank accession No. EU729322) was cloned and expressed. The target gene H.a-ALP, having an open reading frame of 1608 bp, was reverse-transcribed from cDNA by the polymerase chain reaction. The open reading frame of the target gene was cloned into the pET-32a expression vector to obtain recombinant protein in Escherichia coli DE-3 cells for the subsequent production of polyclonal antibody. New Zealand white rabbits were used for production of anti-pET-32a-H.a-ALP. The production of antibody was also optimized by employing ELISA for titer determination. The produced antiserum was processed and used as an antibody. Western blot results showed that the polyclonal antibody produced was capable of effectively binding target protein not only from H. armigera but also from other lepidopterans such as Mythimna separata and Plutella xylostella. This antibody was also used to detect levels of ALP within different instars of H. armigera. Thus, it is concluded that this antibody-based assay is very useful for the effective detection of gene-specific expression. Furthermore, it may also be used to detect the expression levels and tissue localization of ALP, as well as in other physiological studies involving this enzyme.

High-throughput method for ranking the affinity of peptide ligands selected from phage display libraries.

The use of phage display peptide libraries allows rapid isolation of peptide ligands for any target selector molecule. However, due to differences in peptide expression and the heterogeneity of the phage preparations, there is no easy way to compare the binding properties of the selected clones, which operates as a major "bottleneck" of the technology. Here, we present the development of a new type of library that allows rapid comparison of the relative affinity of the selected peptides in a high-throughput screening format. As a model system, a phage display peptide library constructed on a phagemid vector that contains the bacterial alkaline phosphatase gene (BAP) was selected with an antiherbicide antibody. Due to the intrinsic switching capacity of the library, the selected peptides were transferred "en masse" from the phage coat protein to BAP. This was coupled to an optimized affinity ELISA where normalized amounts of the peptide-BAP fusion allow direct comparison of the binding properties of hundreds of peptide ligands. The system was validated by plasmon surface resonance experiments using synthetic peptides, showing that the method discriminates among the affinities of the peptides within 3 orders of magnitude. In addition, the peptide-BAP protein can find direct application as a tracer reagent.

Low expression of antigen-presenting and costimulatory molecules by lung cells from tuberculosis patients

Costimulatory and antigen-presenting molecules are essential to the initiation of T cell immunity to mycobacteria. The present study analyzed by immunocytochemistry, using monoclonal antibodies and alkaline phosphatase-anti-alkaline phosphatase method, the frequency of costimulatory (CD86, CD40, CD40L, CD28, and CD152) and antigen-presenting (MHC class II and CD1) molecules expression on human lung cells recovered by sputum induction from tuberculosis (TB) patients (N = 22) and non-TB controls (N = 17). TB cases showed a statistically significant lower percentage of HLA-DR+ cells than control subjects (21.9 ± 4.2 vs 50.0 ± 7.2 percent, P < 0.001), even though similar proportions of TB cases (18/22) and control subjects (16/17, P = 0.36) had HLA-DR-positive-stained cells. In addition, fewer TB cases (10/22) compared to control subjects (16/17) possessed CD86-expressing cells (P = 0.04; OR: 0.05; 95 percentCI = 0.00-0.51), and TB cases expressed a lower percentage of CD86+ cells (P = 0.04). Moreover, TB patients with clinically limited disease (£1 lobe) on chest X-ray exhibited a lower percentage of CD86-bearing cells compared to patients with more extensive lung disease (>1 lobe) (P = 0.02). The lower expression by lung cells from TB patients of HLA-DR and CD86, molecules involved in antigen presentation and activation of T cells, may minimize T cell recognition of Mycobacterium tuberculosis, fostering an immune dysfunctional state and active TB.

Low expression of antigen-presenting and costimulatory molecules by lung cells from tuberculosis patients.

Costimulatory and antigen-presenting molecules are essential to the initiation of T cell immunity to mycobacteria. The present study analyzed by immunocytochemistry, using monoclonal antibodies and alkaline phosphatase-anti-alkaline phosphatase method, the frequency of costimulatory (CD86, CD40, CD40L, CD28, and CD152) and antigen-presenting (MHC class II and CD1) molecules expression on human lung cells recovered by sputum induction from tuberculosis (TB) patients (N = 22) and non-TB controls (N = 17). TB cases showed a statistically significant lower percentage of HLA-DR+ cells than control subjects (21.9 +/- 4.2 vs 50.0 +/- 7.2%, P < 0.001), even though similar proportions of TB cases (18/22) and control subjects (16/17, P = 0.36) had HLA-DR-positive-stained cells. In addition, fewer TB cases (10/22) compared to control subjects (16/17) possessed CD86-expressing cells (P = 0.04; OR: 0.05; 95%CI = 0.00-0.51), and TB cases expressed a lower percentage of CD86+ cells (P = 0.04). Moreover, TB patients with clinically limited disease ( pound1 lobe) on chest X-ray exhibited a lower percentage of CD86-bearing cells compared to patients with more extensive lung disease (>1 lobe) (P = 0.02). The lower expression by lung cells from TB patients of HLA-DR and CD86, molecules involved in antigen presentation and activation of T cells, may minimize T cell recognition of Mycobacterium tuberculosis, fostering an immune dysfunctional state and active TB.

Placental alkaline phosphatase (PLAP) enzyme activity and binding to IgG in Chagas' disease.

Placentas and plasma from women with and without Chagas' disease and cultures of human placental villi with Trypanosoma cruzi, neuraminidase, phospholipase A2 and phospholipase C were analyzed in order to verify if the alterations in placental alkaline phosphatase (PLAP) enzyme activity are caused by T. cruzi as observed in previous works. As IgG receptivity happens to be one of the proposed functions of PLAP, general IgG binding ability of the placentas treated with the mentioned enzymes, which are present on the parasite's surface, were also tested. The phospholipases caused an increase of PLAP's enzyme activity in the supernatant of infected placentas and a decrease of enzyme activity in the membrane of cultured placentas, therefore suggesting the cleavage of PLAP by parasitic enzymes. Desialylation could also partially inhibit PLAP's enzyme activity in supernatant and membrane of placenta culture. Placentas from healthy patients presented higher IgG receptivity than those from patients with Chagas' disease. In vitro infection of healthy placentas with T. cruzi caused no difference in IgG receptivity in placental sections with respect to controls but the phospholipases and neuraminidase increased the IgG receptivity of cultured placentas. The IgG transference index was higher for patients with Chagas' disease than for those without it. Although binding to IgG does not completely inhibit the enzyme activity of PLAP, it interferes with the enzyme activity of PLAP. We concluded that the enzymes on the surface of T. cruzi trypomastigotes can not only affect PLAP's enzyme activity but also increase the IgG binding ability of the placenta and this can be related to the actions of neuraminidase-transsialidase, phospholipase A2 and phospholipase C on the parasite surface. The modification of PLAP from women with Chagas' disease should be considered as a result of multiple factors.

[Transposon tnPhoA mutagenesis as a method of obtaining mutants of Azospirillum brasilense by motility and flagellation]. / Transpozonovyi tnPhoA mutagenez kak sposob polucheniia mutantov Azospirillum brasilense po podvizhnosti i zhgutikovaniiu.

Three A. brasilense strains (S27, SpBr14, and KR77) did not hydrolyze the chromogenic substrate of alkaline phosphatase (PhoA), X-phosphate, in situ, and were used as recipients in experiments on TnphoA mutagenesis. KMR transconjugates were obtained only for A. brasilense S27, 85% of them were also PhoA+. About 12% TnphoA mutants of A. brasilense S27 had reduces capacity to swarming and 3% of mutants neither swam nor swarmed. These totally immotile clones were examined under transmission electron microscope and were classified as Fla-Laf-, Fla-leakyLaf-, and Fla-Laf+ mutants. In Fla-Laf+ TnphoA mutants of S27, the expression of their lateral flagella (Laf) retained the wild-type inducibility. The presence of intact polar flagellum (Fla) did not seem to be obligatory for controllable expression of Laf in A. brasilense S27. The data suggest that A. brasilense S27 Fla and Laf systems have common structural and/or regulatory components. The PhoA+ phenotype of S27 Fla- mutants suggested a periplasmic and/or membrane localization of the hybrid proteins, the formation of which blocks the flagellar assembly or functioning. Immunochemical analysis with antibodies to alkaline phosphatase will identify these proteins.

Specificity of the solid phase alkaline phosphatase immunocapture assay for the diagnosis of human Schistosoma mansoni infection.

The species specificity of the solid phase alkaline phosphatase immunocapture assay (APIA) for the immunological detection of human immunoglobulin G antibodies to the alkaline phosphatase of adult Schistosoma mansoni was evaluated. Sera from schistosomiasis patients from South America, West Africa, south-east Asia and uninfected control subjects were compared. Only the sera of patients infected with S. mansoni gave positive results. There was no apparent difference between 2 populations infected with S. mansoni, one from South America and the other from West Africa. The results with sera from various regions of West Africa were also indistinguishable. Although the APIA was not able to discriminate the geographical origin of the S. mansoni-infected subjects, the method appeared to be specific for S. mansoni and suitable for use in the immunodiagnosis of schistosomiasis mansoni, particularly in endemic areas where mixed infections of Schistosoma spp. occur.

Parasite enzymes as a tool to investigate immune responses

Previous evidences reported by us and by other authors revealed the presence of IgG in sera of Schistosoma mansoni-infected patients to immunodominant antigens which are enzymes. Besides their immunological interest as possible inductors of protection, several of these enzume antigens might be also intersting markers of infection in antibody-detecting immunocapture assays which use the intrinsic catalytic property of these antigens. It was thus thought important to define some enzymatic and immunological characteristics of these molecules to better exploit their use as antigens. Four different enzymes from adult worms were partially characterized in their biochemical properties and susceptibility to react with antibodies of infected patients, namely alkaline phosphatase (AKP, Mg*+, pH 9.5), type I phosphodiesterase (PDE, pH 9.5), cysteine proteinase (CP, dithiothreitol, pH 5.5) and N-acetyl-ß-D-glucosaminidase (NAG, pH 5.5). The AKP and PDE are distinct tegumental membrane-bound enzymes whereas CP and NAG are soluble acid enzymes. Antibodies in infected human sera differed in their capacity to react with and to inhibit these enzyme antigens. Possibly, the specificity of the antibodies related to the extent of homology between the parasite and the host enzyme might be in part responsible for the above differences. The results are also discussed in view of the possible functional importance of these enzymes

Production of a mouse monoclonal antibody against the alkaline phosphatase of adult Schistosoma mansoni.

This study describes the production and characterization of a monoclonal antibody (mAb) against the alkaline phosphatase of Schistosoma mansoni from splenocytes of chronically infected mice. Convenient selection of the mAb was achieved using the catalytic activity of the antigen in a developed enzyme-antigen immunoassay. The mAb was of the IgG1 subclass and it specifically recognized the alkaline phosphatase in adult worm sections by indirect immunofluorescence. Preincubation of the antibody with partially purified adult alkaline phosphatase did not result in inhibition of the enzyme activity and it did not mediate complement-dependent cytotoxicity against mechanically transformed schistosomula in vitro. The mAb was able to immunoprecipitate under reducing conditions a polypeptide of 65 kDa, similar in size to the monomeric subunit of the schistosome enzyme. The specificity of the mAb was assessed by competitive inhibition with antibodies of infected human sera in an immunoadsorption assay. Periodate treatment of the antigen resulted in altered electrophoretic mobility of alkaline phosphatase, which confirmed the presence of carbohydrate in the molecule, but this did not prevent binding by the mAb. Although the use of the mAb in capture assays for detection of circulating alkaline phosphatase in infected host sera was unsuccessful, the production of this antibody confirmed that the enzyme is exposed by adult worms to the host and that it is immunogenic; additionally, a monoclonal probe is available for further characterization of the structure and function of this important parasite surface molecule.

Antigenicity of adult Schistosoma mansoni alkaline phosphatase.

Antibodies to the alkaline phosphatase (AP) of Schistosoma mansoni in infected human and mice sera were evaluated by a direct solid-phase AP immunoadsorption assay (APIA) and by Western blot and immunostaining. APIA consisted of (a) solid-phase capture of immunoglobulins from infected human or mice, (b) immunoadsorption of the enzyme antigen by the antibodies, and (c) detection of the enzymatic activity. By this procedure the appearance of the anti-AP response in mice was detected around 50 days post-infection; this response was not specific for an AP of a given schistosome strain and it was not induced by an autoimmunity phenomenon. Fourteen out of 15 sera from infected people tested by APIA showed a clear antibody response against this enzyme. Immunoblots in non-reducing conditions supported APIA results indicating that the parasite AP was specifically recognized by the antibodies present in infected human and mice sera. These results suggest the possible usefulness of the schistosome AP as a marker for S. mansoni infection.

Immunodiagnosis of Schistosomiasis mansoni with APIA (alkaline phosphatase immunoassay).

The previously shown antigenicity of Schistosoma mansoni (JL venezuelan strain) alkaline phosphatase (Mg2+, pH 9.5) allowed its use in an immunodiagnosis assay, that consisted in the immunoadsorption of the enzymatic activity. Protein-A coated polyvinyl plates were used as solid phase to capture IgG from sera of infected human patients. After buffered saline washings, the plates were incubated with an enzyme-rich fraction (a n-butanol aqueous extract of adult worm obtained from pairs). Immunoadsorbed alkaline phosphatase (AP) was revealed by adding rho-nitrophenyl phosphate. Anti-AP antibodies were detected in 93% of coproparasitologically proven S. mansoni-infected venezuelan patients but not in parasite-free control sera and sera from patients infected with parasitosis other than schistosomiasis. The APIA did not correlate with cure but the anti-AP antibody response was progressively reduced after treatment. The use of an AP substrate amplifying system allowed an improvement of the assay sensitivity without loss of specificity. The data suggest that the APIA could be used as a marker of infection by S. mansoni.