Serological antibody detection by enzyme-linked immunosorbent assay (ELISA)- and immunoblot-based methods constitutes the best indicator of human Toxocara infection. Nevertheless, the availability of serological tests, particularly western blots (WB), evaluated for sensitivity and specificity is limited. Therefore, an Anti-Toxocara-ELISA immunoglobulin g (IgG) prototype (Proto-ELISA) and an Anti-Toxocara-Westernblot (IgG) prototype (Proto-WB) were evaluated by testing 541 human sera pre-determined for Toxocara infection by an established in-house Anti-Toxocara-ELISA (IH-ELISA). To evaluate sensitivity and specificity of the newly developed ELISA and WB prototypes, results were compared to IH-ELISA and a commercial WB (Com-WB). Compared to the IH-ELISA, a sensitivity of 93.1% (229/246) and a specificity of 94.6% (279/295) of the Proto-ELISA with a Cohen's κ of 0.88 were obtained. The sensitivity of the Proto-WB was 76.7% (240/313) and specificity was 99.6% (227/228) with a Cohen's κ of 0.73 compared to those of Com-WB. A comparison to the IH-ELISA revealed 91.5% (225/246) sensitivity and 94.6% (279/295) specificity of the Proto-WB with a Cohen's κ of 0.86. Cross-reactivity was observed for some samples positive for Ascaris and Trichinella spp. in the Proto-ELISA, Proto-WB and Com-WB. Overall, the evaluated ELISA and WB prototypes showed high sensitivity and specificity, indicating high reliability of these newly developed tests.

Hypoplastic left heart syndrome consists of several structural abnormalities in the left side of the heart and may be associated with a hereditary genetic cause, possibly related to the connexin gene GJA1; however, only a few studies have investigated it. The present study aimed to analyse the expression of connexin-43 in the cardiac muscle of hypoplastic left heart syndrome children by Western blot method and confocal laser scanning microscopy. For that, tissue samples were taken during corrective surgery to treat heart defects. Patients of control group (8) presented any type of heart defect not related to hypoplastic left heart syndrome, connexin-43, or its gene and those of hypoplastic left heart syndrome group (9) presented this disease singly, without any other associated congenital diseases. By means of confocal laser scanning microscopy, it was noticed no connexin-43 qualitative differences in positioning and location pattern between both groups. From Western blot analysis, the connexin-43 expression did not show a statistically significant difference (p = 0.0571) as well. Within the limits of this study, it is suggested that cardiomyocytes of hypoplastic left heart syndrome children are similar in connexin-43 location, distribution, and structural and conformational patterns to those of children with heart defects not related to this protein and its genes.

During follicular development, granulosa cells undergo functional and structural changes affecting their steroidogenic activity. Oestrogen synthesis mainly occurs in the endoplasmic reticulum and relies on aromatase activity to convert androgens that arise from theca cells. In the present study, indicators of mitochondria-related steroidogenic capacity, as steroidogenic acute regulatory (StAR) protein expression and mitochondrial membrane potential (MMP), have been evaluated in bovine granulosa cells (GCs) and related to follicle growth and atresia. Atresia was estimated by morphological examination of follicle walls and cumulus–oocyte complexes (COC) and assessed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay for apoptosis detection. Bovine ovarian follicles were macroscopically classified according to their atresia grade and grouped into small, medium or large follicles. After follicle opening, the COCs were morphologically classified for follicle atresia and the GCs were collected. Granulosa cells were fixed for immunofluorescence (IF) and TUNEL assay, frozen for western blotting (WB) or freshly maintained for MMP analyses. StAR protein expression was assessed using both IF and WB analyses. The follicle atresia grade could be efficiently discriminated based on either follicle wall or COC morphological evaluations. Granulosa cells collected from small non-atretic follicles showed a higher (P <0.01) MMP and WB-based StAR protein expression than small atretic follicles. For IF analysis, StAR protein expression in large atretic follicles was higher (P <0.05) than that in large non-atretic follicles. These results suggest a role played by mitochondria in GC steroidogenic activity, which declines in healthy follicles along with their growth. In large follicles, steroidogenic activity increases with atresia and is possibly associated with progesterone production.

This is the first report on the development and characterization of eight monoclonal antibodies (MABs) generated against whole- and membrane-enriched tachyzoite extracts of the apicomplexan parasite Besnoitia besnoiti. Confocal laser scanning immunofluorescence microscopy was used to localize respective epitopes in B. besnoiti tachyzoites along the lytic cycle. A pattern compatible with dense granule staining was observed with MABs 2.A.12, 2.F.3 and 2.G.4, which could be confirmed by immunogold electron microscopy for MABs 2.A.12 and 2.F.3. In particular, MABs 2.F.3 and 2.G.4 were secreted during early invasion, proliferation and egress phases. MABs 3.10.8 and 5.5.11 labelled the tachyzoite surface, whilst MABs 1.17.8, 8.9.2 and 2.G.A recognized the apical tip, which is reminiscent for microneme localization. Besides, the epitopes recognized by the latter two (MABs 8.9.2 and 2.G.A) exhibited a redistribution from the anterior part across the parasite surface towards the posterior end during invasion. Most MABs developed were genus-specific. Indeed, the MABs cross-reacted neither with T. gondii nor with N. caninum tachyzoites. In summary, we have generated MABs that will be useful to study the key processes in the lytic cycle of the parasite and with additional promising diagnostic value. However, the molecular identity of the antigens recognized remains to be elucidated.

Significant experimental evidence supports fat as a taste modality; however, the associated peripheral mechanisms are not well established. Several candidate taste receptors have been identified, but their expression pattern and potential functions in human fungiform papillae remain unknown. The aim of this study is to identify the fat taste candidate receptors and ion channels that were expressed in human fungiform taste buds and their association with oral sensory of fatty acids. For the expression analysis, quantitative RT-PCR (qRT-PCR) from RNA extracted from human fungiform papillae samples was used to determine the expression of candidate fatty acid receptors and ion channels. Western blotting analysis was used to confirm the presence of the proteins in fungiform papillae. Immunohistochemistry analysis was used to localise the expressed receptors or ion channels in the taste buds of fungiform papillae. The correlation study was analysed between the expression level of the expressed fat taste receptors or ion channels indicated by qRT-PCR and fat taste threshold, liking of fatty food and fat intake. As a result, qRT-PCR and western blotting indicated that mRNA and protein of CD36, FFAR4, FFAR2, GPR84 and delayed rectifying K+ channels are expressed in human fungiform taste buds. The expression level of CD36 was associated with the liking difference score (R −0·567, β=−0·04, P=0·04) between high-fat and low-fat food and FFAR2 was associated with total fat intake (ρ=−0·535, β=−0·01, P=0·003) and saturated fat intake (ρ=−0·641, β=−0·02, P=0·008).

In the present study, we analyse the effect of glycosylation in Echinostoma caproni (Trematoda: Echinostomatidae) antigens in antibody responses against the parasite in experimentally infected mice. It has been previously demonstrated that the mouse is a host of high compatibility with E. caproni and develops elevated responses of IgG, IgG1, IgG3 and IgM as a consequence of the infection, though the role of glycans in these responses remains unknown. To this purpose, the responses generated in mice against non-treated excretory/secretory antigens of E. caproni were compared with those observed after N-deglycosylation, O-deglycosylation and double deglycosylation of the antigens by indirect ELISA and western blot. Our results suggest that E. caproni-expressed glycans play a major role in the modulation of the immune responses. The results obtained indicate that IgG subclass responses generated in mice against E. caproni are essentially due to glycoproteins and may affect the Th1/Th2 biasing. The reactivity significantly decreased after any of the deglycosylation treatments and the N-glycans appears to be of greater importance than O-glycans. Interestingly, the IgM response increased after N-deglycosylation suggesting that carbohydrates may mask peptide antigens.

To assess the prevalence of Taenia solium cysticercosis in patients with neurological disorders in Slovenia, serum/cerebrospinal fluid (CSF) samples from 348 suspected patients were collected between the beginning of January 2001 and the end of December 2012 and analysed serologically for the presence of anti-T. solium IgG antibodies. Of 20 patients whose samples tested positive or equivocal by enzyme-linked immunosorbent assay (ELISA), samples of 7 patients were confirmed positive by Western blot (WB). The overall seroprevalence rate of T. solium infection in patients with neurological disorders included in the study was 2.0%. Serological results of positive patients corresponded to clinical and/or imaging findings concerning their brain cysts. Based on their personal data, it was ascertained that neurocysticercosis (NCC) positive patients had immigrated or came to Slovenia from the former Yugoslav republics. Since the disease is believed not to be endemic in Slovenia we assume that all of the NCC-positive patients had acquired the infection before immigration to Slovenia or visiting or being visited by their relatives infected with an adult T. solium parasite. The present results represent the first insight into the prevalence of NCC in patients with neurological disorders in Slovenia.

The objective of this work was to investigate the expression of heat shock protein 70 (HSP70) by Western blot (WB) in swine liver. Subsequently, the study aimed to apply this method to two experimental groups of heavy pigs raised in different confinement systems: intensive/indoor (Group A) and extensive/outdoor (Group B). Thirty-six crossbred commercial heavy pigs were divided as follows: Group A (eight castrated males and eight females) was equally distributed into two single-sex indoor pens (1.02 m2/pig); Group B (11 castrated males and nine females) was kept in one single (partially grassy and partially wooded) open area of about 6000 m2. Group A was slaughtered at 41 weeks of age (170 ± 9 kg) and Group B at 48 weeks of age (172 ± 13 kg). At the abattoir the livers of all the animals were collected and analyzed by WB assay in order to quantify the levels of HSP70. Moreover, a further liver sample was taken from the same animals in order to investigate the cellular localization of HSP70 by immunohistochemistry (IHC). The interaction between sex and group resulted statistically significant (P = 0.001). When stratified by sex, Group A showed significantly higher HSP70 values compared with Group B for both male and female subjects (P < 0.001). Stratifying by group, males showed significantly higher HSP70 values than females in Group A (P < 0.001), whereas no statistical differences were observed between sexes for Group B (P = 0.653). The IHC results evidenced cytoplasmic immunoreactivity in a granular pattern in both groups. The different expression pattern observed by WB could prove to be a useful tool in the assessment of pig health and welfare.

The pancreatic enzyme secretory phospholipase A2 group IB (sPLA2 IB) hydrolyses phospholipids at the sn-2 position, resulting in a NEFA and a lyso-phospholipid, which are then absorbed by the enterocytes. The sPLA2 IB is a member of a family of nineteen enzymes sharing the same catalytic ability, of which nine are cytosolic and ten are secretory. Presently, there are no pharmacological tools to separate between the different secretory enzymes when measuring the enzymatic activity. Thus, it is important to support activity data with more precise techniques when isolation of intestinal content is not possible for analysis, as in the case of small teleost larvae, where the whole animal is sometimes analysed. In the present study, we characterise the sPLA2 IB gene in Atlantic cod (Gadus morhua) and describe its ontogeny at the genetic and protein level and compare this to the total sPLA2 activity level. A positive correlation was found between the expression of sPLA2 IB mRNA and protein. Both remained stable and low during the larval stage followed by an increase from day 62 posthatch, coinciding with the development of the pyloric ceaca. Meanwhile, total sPLA2 enzyme activity in cod was stable and relatively high during the early stages when larvae were fed live prey, followed by a decrease in activity when the fish were weaned to a formulated diet. Thus, the expression of sPLA2 IB mRNA and protein did not correlate with total sPLA2 activity.

The serum antibody response (total, and isotypes IgG1, IgG4, IgM, IgA and IgE) to Guinea worm infection was examined in humans from a highly endemic area of northern Ghana by ELISA and SDSPAGE/Western blot techniques using an adult D. medinensis antigen. Sera were obtained early and late in the peak transmission period, from persons with patent and postpatent infections, as well as from persons from the same endemic area who claimed never to have had Guinea worm infection. To observe for potential cross-reactions in the tests, sera were also obtained from areas with no transmission of Guinea worm from patients with hookworm. O. volvulus and W. bancrofti infections, and from non-infected controls. Sera from persons living in the Guinea worm endemic area reacted extensively with Guinea worm antigen in both tests, and large numbers of bands were produced in the Western blots (up to 35 identified for some sera). For most antibody isotypes, the ELISA absorbance values obtained with sera from the same individuals varied between the two transmission seasons, with the highest titres present towards the end of the peak transmission period. The mean antibody titres for persons in the patent and postpatent infection categories were not significantly different when sera were obtained at the same season of the year. Persons from the endemic area, who claimed never to experience patent infections, also had antibodies to Guinea worm, although at significantly lower mean levels than for the patent and postpatent categories. The highest specificity in the ELISA and the most homogenous Western blots were obtained when detecting for antibodies of the IgG4 isotype.

The serum antibody response in golden hamsters (Mesocricetus auratus) infected with the intestinal trematode Echinostoma caproni was examined with ELISA, SDS-PAGE and Western blot, and IFAT techniques. All methods showed that the hamsters responded slowly but developed a clear positive humoral response to the infection. In most hamsters, an antibody response to infection could not be detected earlier than 11–13 weeks after infection with 6 or 25 metacercariae, and responses were weak when compared to previous results from mice infected with the same parasite. IFAT with positive hamster sera on live juvenile E. caproni showed only fluorescence at the posterior tip, which is a different pattern from that seen using sera from infected mice, indicating a different response to antigens on the juvenile parasites by these two hosts. The results are discussed in relation to the limited selfcure and development of resistance which is observed in golden hamsters infected with E. caproni.

In human Toxocara canis infection, an association has been shown between high IgG avidity in the chronic phase and low IgG avidity in recently acquired toxocarosis. The evolution of the antibody response in terms of avidity has been carried out through a T. canis infection in BALB/c mice. Infection with T. canis embryonated eggs (EE) was carried out with single doses (SD) of 6, 12, 50, 100, 200 or 1000 EE/mouse and with multiple doses (MD) of 200 and 1000 EE. Specific antibodies against T. canis (IgM+G, IgG, IgG1 and IgM) were detected by ELISA and Western Blot (WB) techniques in the presence and absence of urea. With the ELISA method, an increase in the avidity index (AI) of around 50% was detected from days 40–80 p.i. to the end of the study, with all the doses studied. The WB method showed the presence of high avidity antibodies bound to 100 kDa and 75 kDa T. canis proteins in all the cases when the IgM+G and the IgG1 antibodies were investigated. Antibodies of variable avidity were observed in those sera that recognized the group of low molecular weight proteins, between 37 kDa and 25 kDa.

Toxocariasis is a common helminthic infection that has a worldwide distribution. However, data from Lebanon about the prevalence of this infection are non-existent. We conducted a Toxocara seroprevalence study with 150 subjects attending the outpatient clinics at the American University of Beirut Medical Center between May and June 2004. Serum specimens were tested for anti-Toxocara antibodies by enzyme-linked immunosorbent assay and confirmed by Western blot. Multivariate analysis was performed to identify risk factors for infection. The seroprevalence rate of toxocariasis was 19%. Male gender and below high school education were significantly associated with a positive serological test (odds ratios=3·1 and 2·8, respectively). Higher numbers of persons in the household, and low family income during childhood, were significant on bivariate analysis only. Toxocariasis is common in Lebanon. A large population-based survey is needed to confirm these results.

Previous studies have demonstrated that peripheral inflammation leads to long-term increases in spinal dorsal horn neuronal excitability (i.e. central sensitization) and behavioral hyperalgesia that is mediated by glutamate receptor activation. The present study has examined spinal cord changes in serine phosphorylation of the NMDA receptor NR1 and AMPA receptor GluR1 subunits after peripheral inflammation. Using site-specific antibodies, Western blots showed a significant increase in phosphorylation at serine residues 896 and 897 of the NR1 and at serine residues 831 and 845 of the GluR1 subunits in the rat spinal cord after injection of complete Freund's adjuvant into the hindpaw. For all four serine sites, the increased phosphoserine NR1 and GluR1 levels occurred as early as 10 minutes, lasted for at least 3 days, and returned to control level 7–14 days following inflammation. There was also an increase in total NR1 and GluR1 proteins after inflammation, but with a different time course. The phosphorylation of different serine residues of NR1 and GluR1 depended on input from the injured site. Local anesthesia of the inflamed paw with lidocaine (2%), blocked the increase in phosphoserine 896 (NR1) and phosphoserine 831 (GluR1) with a time course corresponding to the duration of anesthesia. However, the increased phosphoserine 845 (GluR1) was blocked only transiently and the concentration of phosphoserine 897 (NR1) was not affected by local anesthesia. Consistent with previous biochemical analysis, phosphorylation of serine 896 NR1 and serine 831 of GluR1 was blocked by intrathecal chelerythrine (0.01–1 nmol), a PKC inhibitor, and phosphorylation of serine 897 NR1 was blocked by H-89 (0.01–1 nmol), a PKA inhibitor. In addition, there appeared to be an interaction between the PKC and PKA cellular pathways because phosphorylation of serine 897 of NR1, a substrate of PKA, was also blocked by chelerythrine. These findings correlate serine phosphorylation of the NR1 and GluR1 subunits to spinal hyperexcitability and hyperalgesia, and indicate a role of serine phosphorylation of NMDA and AMPA receptors in initiating central sensitization.

Small nuclear ribonucleoproteins (snRNPs) are involved in trans-splicing processing of pre-mRNA in Trypanosoma cruzi. To clone T. cruzi snRNPs we screened an epimastigote cDNA library with a purified antibody raised against the Sm-binding site of a yeast sequence. A clone was obtained containing a 507 bp-insert with an ORF of 399 bp and coding for a protein of 133 amino acids. Sequence analysis revealed high identity with the L27 ribosomal proteins from different species including: Canis familiaris, Homo sapiens, Schizosaccharomyces pombe and Saccharomyces cerevisiae. This protein has not been previously described in the literature and seems to be a new ribosomal protein in T. cruzi and was given the code TcrL27. To express this recombinant T. cruzi L27 ribosomal protein in E. coli, the insert was subcloned into the pET32a vector and a 26 kDa recombinant protein was purified. Immunoblotting studies demonstrated that this purified recombinant protein was recognized by the same anti-Sm serum used in the library screening as well as by chagasic and systemic lupus erythemathosus (SLE) sera. Our results suggest that the T. cruzi L27 ribosomal protein may be involved in autoimmunity of Chagas disease.

Many viruses have overlapping genes and/or regions in which a nucleic acid signal is embedded in a coding sequence. To search for dual-use regions in the hepatitis C virus (HCV), we developed a facile computer-based sequence analysis method to map dual-use regions in coding sequences. Eight diverse full-length HCV RNA and polyprotein sequences were aligned and analyzed. A cluster of unusually conserved synonymous codons was found in the core-encoding region, indicating a potential overlapping open reading frame (ORF). Four peptides (A1, A2, A3, and A4) representing this alternate reading frame protein (ARFP), two others from the HCV core protein, and one from bovine serum albumin (BSA) were conjugated to BSA and used in western blots to test sera for specific antibodies from 100 chronic HCV patients, 44 healthy controls, and 60 patients with non-HCV liver disease. At a 1:20,000 dilution, specific IgGs to three of the four ARFP peptides were detected in chronic HCV sera. Reactivity to either the A1 or A3 peptides (both ARFP derived) was significantly associated with chronic HCV infection, when compared to non-HCV liver disease serum samples (10/100 versus 1/60; p < 0.025). Antibodies to A4 were not detected in any serum sample. Our western blot assays confirmed the presence of specific antibodies to a new HCV antigen encoded, at least in part, in an alternate reading frame (ARF) overlapping the core-encoding region. Because this novel HCV protein stimulates specific immune responses, it has potential value in diagnostic tests and as a component of vaccines. This protein is predicted to be highly basic and may play a role in HCV replication, pathogenesis, and carcinogenesis.