A competitive enzyme-linked immunosorbent assay for rapid detection of antibodies against Trichinella spiralis and T. britovi - one test for humans and swine.

Infection with parasites from the Trichinella genus occurs in many vertebrates but disease only occurs in humans (trichinellosis). Humans are infected after the consumption of raw or undercooked meat from infected wild or domestic animals (usually swine or horses). Using the monoclonal antibody (mAb) 7C2C5, specific for an epitope unique to the muscle larvae of the genus Trichinella, we have developed a competitive enzyme-linked immunosorbent assay (c-ELISA) that enables the rapid detection of Trichinella-specific antibodies in sera originating from two different host species (human, swine) infected with either Trichinella spiralis or Trichinella britovi. This novel c-ELISA exhibited 100% specificity and sensitivity, as confirmed by a Western blot test. The assay is easy to use (one incubation step), and the time required for the procedure (45 min) is shorter than in any other ELISA format. This test could be useful for both the detection and surveillance of Trichinella infections.

The influence of TNF alpha -308 G/A polymorphism on oxidative stress in patients with chronic lymphocytic leukemia.

The aim of this study was to examine the association of TNF-α-308G/A polymorphism with CLL and influence on oxidative stress parameters.Significant difference in the genotype and allele distribution was obtained in TNFA subgroup of patients.Significantly higher GPx activity and TBARS and lower catalase activity were detected in CLL.Significantly higher catalase and lower GPx activities were detected in PBMC of TNFG compared to TNFA subgroup, while TBARS were higher in TNFA.Oxidative stress in CLL patients highly correlates with the presence of TNFA subgroup. Increased TBARS, GPx and decreased catalase activity are associated with TNF-α-308A allele containing genotypes.

Immunomodulatory potential of particular Trichinella spiralis muscle larvae excretory-secretory components.

Excretory-secretory antigens of Trichinella spiralis muscle larvae can induce the semi-matured status of rat dendritic cells. This may at least partly be the consequence of transient activation of extracellular signal-regulated kinases 1/2 (ERK1/2). Here we investigated the potential of several components of excretory-secretory antigens (native fraction containing 45, 49 and 53kDa proteins and recombinant Tsp53, representing one of the constituents of this fraction) to demonstrate previously observed effects of excretory-secretory antigens on dendritic cells in vitro, characterised by establishment of a particular phenotype (very low MHC II expression, moderate CD86 expression and significant ICAM-1 expression) and functional properties (low production of pro-inflammatory cytokine IL-12p70, and high production of IL-10 and TGF-ß). Dendritic cells activated by these components were able to provoke proliferation of naïve T cells and their polarisation towards Th2 and anti-inflammatory responses. The investigated antigens had almost the same capacity to induce IL-4 and IL-10 production from T cells as excretory-secretory antigens, but failed to induce significant TGF-ß synthesis. It could be concluded that the investigated excretory-secretory antigens components can largely reproduce the immunomodulatory effects of the complete excretory-secretory antigens and therefore may be considered as molecules important for creation of the anti-inflammatory milieu achieved by the parasite.

Trichinella infections in different host species of an endemic district of Serbia.

Trichinella infections are endemic in the Balkan region of Europe. Though trichinellosis and agents thereof are serious problems for human health and animal husbandry, only a limited number of Trichinella isolates from Serbia have been identified at the species level so far. The aim of the present study was the surveillance and monitoring of Trichinella in domestic pigs and wild animals from the endemic district of Branicevo. Investigations performed during the 2009-2010 period revealed Trichinella infections in 344 out of 282,960 (0.12%) domestic pigs. Among wildlife, Trichinella infections were detected in 11 out of 94 (11.7%) wild boars (Sus scrofa), 7 out of 57 (12.3%) red foxes (Vulpes vulpes), 7 out of 13 (53.8%) golden jackals (Canis aureus), and in all three examined wolves (Canis lupus). Trichinella spiralis and Trichinella britovi were the only two species identified. T. britovi was identified in 31% of isolates from wildlife of the Branicevo district and T. spiralis was found in 53% of wild animals; mixed infections were observed in 16% of the animals examined. Findings form the basis of an information campaign for veterinary services, pig owners and the hunter's associations about the risk of the transmission of these zoonotic agents. The application of control programs as established at the Veterinary Specialist Institute of Pozarevac resulted in a decline in Trichinella infections among domestic pigs and the absence of human trichinellosis in the last three years in the Branicevo district.

Application of dendritic cells stimulated with Trichinella spiralis excretory-secretory antigens alleviates experimental autoimmune encephalomyelitis.

The parasitic nematode, Trichinella spiralis (T. spiralis), exerts an immunomodulatory effect on the host immune response through excretory-secretory products (ES L1) released from encysted muscle larvae. Our model of combined T. spiralis infection and experimental autoimmune encephalomyelitis (EAE) in Dark Agouti (DA) rats demonstrated a significant reduction in EAE severity in infected animals. Recently, we have created an immune status characteristic for the live infection by in vivo application of dendritic cells (DCs) stimulated with ES L1 products of T. spiralis muscle larvae. Moreover, these cells were able to ameliorate EAE when applied 7 days before EAE induction. ES L1-stimulated DCs increased production of IL-4, IL-10 and TGF-ß, and decreased production of IFN-γ and IL-17, both at the systemic level and in target organs. A significant increase in the proportion of CD4+CD25+Foxp3+ T cells was found among spleen cells, and CNS infiltrates from DA rats treated with ES L1-stimulated DCs before EAE induction, compared to controls injected with unstimulated DCs. Regulatory T cells, together with elevated levels of IL-10 and TGF-ß, are most likely involved in restraining the production of Th1 and Th17 cytokines responsible for autoimmunity and thus are responsible for the beneficial effect of ES L1-educated DCs on the course of EAE. Our results show that ES L1 antigen-stimulated DCs are able not only to provoke, but also to sustain anti-inflammatory and regulatory responses regardless of EAE induction, with subsequent amelioration of EAE, or even protection from the disease.

Response to oyster mushroom (Pleurotus ostreatus) extract by sensitized and nonsensitized guinea pig trachea.

Occupational exposure to oyster mushroom (Pleurotus ostreatus) has been associated with obstructive lung disease. Previously, we studied an extract of oyster mushroom (OME) and determined that it causes dose-dependent contractions of nonsensitized guinea pig trachea (GPT). We extend these studies to the investigation of sensitized tissue. In the present study 24 animals were sensitized using ovalbumin (OA) and subsequently challenged with an aerosol of 2.5% OA. A control group of 12 nonsensitized GPs was also studied. Tracheas were removed and were divided into rings in which the epithelium was retained (EP+) or removed (EP-). Dose-related contractions of sensitized and nonsensitized GPTs were elicited with OME. In nonsensitized animals the EP+ GPTs demonstrated a significantly greater response to OME (100-1000 µl) than did the EP- GPTs (p < 0.01). By contrast, in sensitized GPTs with and without epithelium there was no difference to challenge with OME. Finally, sensitized GPTs with and without epithelium and nonsensitized GPTs with epithelium responded similarly to challenge with OME. These findings suggest that in nonsensitized animals there is an enhancement of contractile response to OME which is in part mediated by the GPT epithelium. In sensitized animals with or without epithelium, the level of bronchoconstrictor response is similar to that of the nonsensitized animals with epithelium, suggesting an enhanced constrictor response independent of epithelium in the sensitized animals.

Allelic polymorphisms in the repeat and promoter regions of the interleukin-4 gene and malaria severity in Ghanaian children.

Immunoglobulin E has been associated with severe malaria suggesting a regulatory role for interleukin (IL)-4 and/or IgE in the pathogenesis of severe malaria. We have investigated possible associations between polymorphisms in the IL-4 repeat region (intron 3) and promoter regions (IL-4 +33CT and - 590CT) in Ghanaian children with severe malaria. There was a significantly higher frequency of IL-4 intron-3 B1B1 genotype in the cerebral malaria group [P < 0.0001, odds ratio (OR) = 8.7]. The genotype and allele frequencies of the IL-4 -590 and +33 polymorphisms did not differ between the four study groups. Carriers of IL-4 +33T/-590T with cerebral malaria had elevated total IgE compared to non-carriers (P = 0.03). Our data suggest that IL-4 and/or IgE play a regulatory role in the pathogenesis of severe or complicated malaria.

Polymorphisms in interleukin-1beta and interleukin-1 receptor antagonist genes and malaria in Ghanaian children.

We have investigated the possible associations between polymorphisms in two interleukin-1 (IL-1) genes and severity of Plasmodium falciparum malaria in Ghanaian children with cerebral malaria, severe anaemia or uncomplicated malaria and controls. There was no significant difference in genotype and allele frequencies in IL-1beta exon 5 or interleukin-1 receptor antagonist (IL-1ra) polymorphisms between the studied groups, suggesting that the two polymorphisms may not be involved in the pathogenesis of severe malaria. When parasitaemias in uncomplicated malaria patients were evaluated, a significantly higher level of parasitaemia was observed among carriers of IL-1beta A2 allele as compared with noncarriers of this allele (P = 0.01). The mean parasitaemia in an age-matched asymptomatic group did not reveal such associations. These data suggest that IL-1beta exon 5 allele 2 may play a possible role in the clinical outcome of uncomplicated malaria.

Increased levels of autoantibodies against copper-oxidized low density lipoprotein, malondialdehyde-modified low density lipoprotein and cardiolipin in patients with rheumatoid arthritis.

OBJECTIVES: To analyse the association of autoantibodies against cardiolipin (CL) and oxidized low density lipoproteins [copper-oxidized low density lipoprotein (oxLDL), malondialdehyde-modified LDL (MDA-LDL)] with rheumatoid arthritis (RA) and cardiovascular complications. METHODS: One hundred and twenty-one patients with RA were consecutively included. Autoantibodies were determined by ELISA. Healthy individuals from the same region were used as controls. RESULTS: Levels of IgG, IgM and IgA antibodies against MDA-LDL and CL, as well as IgG and IgA antibodies against oxLDL were increased in the patients (P<0.01). The prevalence of IgG, IgM and IgA antibodies against CL was higher than in the normal population (74, 82 and 14%, respectively). The prevalence of IgG and IgA antibodies against oxLDL was also significantly increased (35 and 25%, respectively) and so was the prevalence of IgG and IgM antibodies against MDA-LDL (17 and 26%, respectively) compared with controls. The levels of IgM and IgA antibodies against aCL and IgM against MDA-LDL were increased in patients with extra-articular manifestations. Patients who developed myocardial infarction had a higher prevalence of IgG antibodies against MDA-LDL (P=0.04). There were substantial correlations between the levels of antibodies against oxLDL, MDA-LDL and CL. CONCLUSIONS: RA patients had increased levels and prevalence of autoantibodies against CL, oxLDL and MDA-LDL, with associations to severity of disease and cardiovascular complications.

Nickel and strontium nitrates as modifiers for the determination of selenium in wine by Zeeman electrothermal atomic absorption spectrometry.

A mixed matrix modifier of nickel and strontium nitrates was used as a chemical modifier for the determination of selenium in wines by Zeeman electrothermal atomic absorption spectrometry. Wine samples were heated on a boiling water bath with small amounts of nitric acid and hydrogen peroxide. For complete elimination of interference, especially from sulfates and phosphates, selenium is complexed with ammonium pyrolidinedithiocarbamate (APDTC), extracted into methyl isobutyl ketone (MIBK), and measured by ETAAS. The graphite furnace temperature program was optimized for both aqueous and organic solutions. Pyrolysis temperatures of 1300 degrees C and 800 degrees C were chosen for aqueous and organic solutions, respectively; 2700 degrees C and 2100 degrees C were used as optimum atomization temperatures for aqueous and organic solutions, respectively. The optimum modifier mass established is markedly lower than those presented in the literature. The platform atomization ensures pretreatment stabilization up to 1100 degrees C and 1600 degrees C, respectively, for organic and aqueous selenium solutions. The procedure was verified by the method of standard addition. The investigated wine samples originated from the different regions of the Republic of Macedonia. The selenium concentration varied from not detectable to 0.93 microg L(-1).