Anti-Anopheles darlingi saliva antibodies as marker of Plasmodium vivax infection and clinical immunity in the Brazilian Amazon

Background: Despite governmental and private efforts on providing malaria control, this disease continues to be a major health threat. Thus, innovative strategies are needed to reduce disease burden. The malaria vectors, through the injection of saliva into the host skin, play important role on disease transmission and may influence malaria morbidity. This study describes the humoral immune response against Anopheles (An.) darlingi saliva in volunteers from the Brazilian Amazon and addresses the association between levels of specific antibodies and clinical presentation of Plasmodium (P.) vivax infection. Methods: Adult volunteers from communities in the Rondonia State, Brazil, were screened in order to assess the presence of P. vivax infection by light microscopy and nested PCR. Non-infected volunteers and individuals with symptomatic or symptomless infection were randomly selected and plasma collected. An. darlingi salivary gland sonicates (SGS) were prepared and used to measure anti-saliva antibody levels. Plasma interleukin (IL)-10 and interferon (IFN)-gamma levels were also estimated and correlated to anti-SGS levels. Results: Individuals infected with P. vivax presented higher levels of anti-SGS than non-infected individuals and antibody levels could discriminate infection. Furthermore, anti-saliva antibody measurement was also useful to distinguish asymptomatic infection from non-infection, with a high likelihood ratio. Interestingly, individuals with asymptomatic parasitaemia presented higher titers of anti-SGS and lower IFN-gamma/IL-10 ratio than symptomatic ones. In P. vivax-infected asymptomatic individuals, the IFN-gamma/IL-10 ratio was inversely correlated to anti-SGS titers, although not for while in symptomatic volunteers. Conclusion: The estimation of anti-An. darlingi antibody levels can indicate the probable P. vivax infection status and also could serve as a marker of disease severity in this region of Brazilian Amazon.

Clinicopathological evaluation of in vivo epidermal nuclear fluorescence

Nuclear fluorescence in keratinocytes is an occasional phenomenon, often present in autoimmune diseases, especially in connective-tissue disease (CTD); however, its clinical significance remains unclear. To investigate the profile of patients with positive nuclear staining on direct immunofluorescence (DIF) of skin samples. A retrospective analysis of 28 patient records from our immunodermatology laboratory was performed between May 2003 and June 2006. Inclusion criteria were the presence of autoantibodies (IgG, IgA or IgM) or complement (C3) binding keratinocyte nuclei on DIF. The most prevalent diseases related to the nuclear keratinocyte DIF staining were systemic lupus erythematosus (n = 9), mixed CTD (n = 3), overlap syndrome (n = 3), Sjogren`s syndrome (n = 1), and CREST (calcinosis, Raynaud`s phenomenon, oesophageal dysmotility, sclerodactyly and telangiectasia) syndrome (n = 1). Serum antinuclear antibody (ANA) was positive in 20 of 28 patients, with titres varying from 1 : 160 to 1 : 1280. Of the 20 patients with positive anti-nuclear antibodies (ANA), 17 were positive for anti-extractable nuclear antigen antibodies, 12 had anti-SSA/Ro, 11 had anti-SSB/La and 8 had anti-ribonucleoprotein. Eight patients were negative for ANA. Positive predictive value of in vivo ANA for systemic CTDs was 75%. The present data suggest that in vivo ANA evaluation is an additional and feasible auxiliary tool for diagnosing CTDs.

Anti-C1q Antibodies in Juvenile-Onset Systemic Lupus Erythematosus

The objective of this study was to evaluate the presence of anti-C1q antibodies Hospital Israelita Albert Einstein Research Institute, Sao Paulo, Brazil in 67 juvenile Systemic lupus erythematosus (JSLE) patients and 26 healthy controls and to assess the association of these antibodies with disease activity, nephritis, and presence of anti-double-stranded (ds)DNA. Anti-C1q antibodies were detected by ELISA. A higher frequency of anti-C1q antibodies was observed in JSLE patients compared to controls (20% vs. 0%, P = 0.016). Specificity of these antibodies was 100% [95% confidence interval (CI) 86.7-100%] and sensitivity was 19.4% (95% CI 10.7-30.8%) for a lupus diagnosis. The median anti-C1q antibodies was higher in JSLE patients compared to controls [median (range) 9.4 (5.5-127) vs. 7.3 (5-20) units, P = 0.004]. Remarkably, a positive Spearman`s coefficient was found between anti-dsDNA and anti-C1q units (r = 0.42, P = 0.0004, 95% CI 0.19-0.60). Our results confirm a low frequency of anti-C1q antibody in our lupus populations, but the presence of anti-C1q antibodies appears to be a good marker for JSLE diagnosis.

Novel humanized anti-CD3 antibodies induce a predominantly immunoregulatory profile in human peripheral blood mononuclear cells

Strategies to minimize the immunogenicity and toxicity of murine anti-CD3 antibodies (e.g. OKT3) are of special interest for organ transplantation and for the treatment of autoimmune diseases. In the present work, we have developed two humanized anti-CD3 antibodies. These molecules were shown to bind to human CD3, though less efficiently, and display less mitogenic activity than CKT3. These results prompted us to investigate whether this reduced mitogenic potential was associated with the development of anti-inflammatory properties. Indeed, in peripheral blood mononuclear cells (PBMCs), the humanized antibody versions induced a predominantly anti-inflammatory cytokine profile, in contrast with the pro-inflammatory profile induced by OKT3. Neither OKT3 nor the humanized versions induced the expression of IL-4, IL-2 or TGF-beta. Both humanized antibodies induced significantly lower production of IFN-gamma and IL-5 and slightly higher production of IL-10 than OKT3. This immunomodulatory profile was most evident by the 80-fold higher ratio of IL-10/IFN-gamma production in PBMCs cultured in the presence of the humanized antibodies, compared to those stimulated with CKT3. Furthermore, these humanized anti-CD3 antibodies induced a late FOXP3 gene expression while OKT3 led to a more transient expression of FOXP3. Taken our results, we suggest that these humanized anti-CD3 antibodies may promote the development of T cells with immunoregulatory activity. (C) 2009 Elsevier B.V. All rights reserved.

The frequency of anti-beta(2)-glycoprotein I antibodies is low and these antibodies are associated with pulmonary hypertension in mixed connective tissue disease

The objective of this study is to evaluate the prevalence of antiphospholipid antibodies, mainly anti-beta(2)-glycoprotein I (anti-beta(2)-GPI), and their possible clinical and laboratory relevance in mixed connective tissue disease (MCTD). This study included 39 consecutive patients with MCTD (Kasukawa`s criteria) from January, 2005, to March, 2007, and compared them with 21 age- and sex-matched healthy controls. IgG and IgM anticardiolipin (aCL) and anti-beta(2)-GPI were measured by ELISA. Lupus anticoagulant (LA) was detected by functional coagulation tests. Medium to high titres of aCL and anti-beta(2)-GPI antibodies were found in sera from four (10.2%) MCTD patients. One of these patients was found to be positive for IgM aCL, IgM anti-beta(2)-GPI and LA antibodies simultaneously. Additionally, this patient had a previous history of foetal loss in the second trimester and new-onset pulmonary arterial hypertension (PAH). The other three patients had none of the manifestations of antiphospholipid syndrome (APS) or PAH. The mean value of IgG anti-beta(2)-GPI was higher among those MCTD patients with PAH than in the group without PAH (34.2 +/- 46.8 vs 12.3 +/- 9.1, P = 0.018). None of the controls were positive for antiphospholipid antibodies. High to moderate titres of anti-beta(2)-GPI as well as APS were rare in MCTD, and these antibodies may be correlated with the development of PAH in these patients. Lupus (2009) 18, 618-621.

Val247Leu polymorphism of beta(2) glycoprotein 1 gene may justify the genesis of anti beta(2)GP1 antibodies and Antiphospholipid Syndrome in Multibacillary Leprosy

BACKGROUND - Multibacillary (MB) leprosy may be manifested with antiphospholipid antibodies (aPL), among which anti-beta(2)GP1 (beta(2)-glycoprotein 1). High titers of aPL are associated with APS (Antiphospholipid Syndrome), characterized by thrombosis. The mutation Val247Leu in the domain V of beta(2)GP1 exposes hidden epitopes with consequent development of anti-beta(2)GP1 antibodies. OBJECTIVE: To evaluate the Val247Leu polymorphism of beta(2)GP1 gene and its correlation with anti-beta(2)GP1 antibodies in leprosy patients. METHODS: The Val247Leu polymorphism was performed by PCR-RFLP and anti-beta(2)GP1 antibodies were measured by ELISA. RESULTS: The genotypic Val/Val was more prevalent in the leprosy group, compared to controls. Regarding the 7 MB patients with APS, four presented heterozygosis and three, Val/Val homozygosis. Although higher titrations of anti-beta(2)GP1 IgM antibodies were seen in MB leprosy group with Val/Leu and Val/Val genotypes, there was no statistical difference when compared to Leu/Leu genotype. CONCLUSION: The prevalence of Val/Val homozygosis in leprosy group can partially justify the presence of anti-beta(2)GP1 IgM antibodies in MB leprosy. The description of heterozygosis and Val/Val homozygosis in 7 patients with MB leprosy and thrombosis corroborates the implication of anomalous phenotype expression of beta(2)GP1 and development of anti-beta(2)GP1 antibodies, with consequent thrombosis and APS.

Anti-nucleosome and anti-chromatin antibodies are present in active systemic lupus erythematosus but not in the cutaneous form of the disease

The objective of this study is to investigate the presence of anti-nucleosome (anti-NCS) and anti-chromatin (anti-CRT) antibodies in patients with cutaneous lupus erythematosus (CLE) compared with active and inactive systemic lupus erythematosus (SLE). A total of 154 subjects were evaluated: 54 patients presenting CLE, 66 patients with active SLE and 34 with inactive SLE. Lupus activity was assessed using the disease activity index (SLEDAI). Anti-NCS and anti-CRT antibodies were detected by enzyme-linked immunosorbent assay ( ELISA). Only one of 54 patients with CLE tested positive for both anti-NCS and anti-CRT antibodies. The prevalence of anti-CRT antibodies was significantly higher in active SLE (84.8%) when compared with inactive SLE (26.4%) and CLE (1.8%) ( P < 0.001). Anti-NCS antibodies were also more prevalent in active SLE patients (74.2%) than inactive SLE (11.7%) and CLE patients ( 1.8%) ( P < 0.001). The presence of anti-CRT and anti-NCS antibodies was correlated to disease activity in patients with SLE (r = 0.4937, r = 0.5621, respectively). Furthermore, the detection of both antibodies was correlated with disease activity in patients with SLE who tested negative for anti-dsDNA antibodies ( r = 0.4754 for anti-NCS and r = 0.4281 for anti-CRT). The presence of these two auto-antibodies was strongly associated with renal damage in patients with SLE ( OR = 13.1, for anti-CRT antibodies and OR = 25.83, for anti-NCS antibodies). The anti-NCS and anti-CRT antibodies were not found in CLE. In patients with SLE, there is a correlation of these antibodies with disease activity and active nephritis. When compared with anti-dsDNA antibodies, anti-NCS and anti-CRT antibodies were more sensitive in detecting disease activity and kidney damage in lupus patients. Lupus (2009) 18, 223-229.

Inquiry of antibodies anti-Neospora caninum in dairy cattle, dogs and rural workers of the south-west region of Mato Grosso State

Considering the importance of neosporosis in the animal health and production, the frequency of antibodies to Neospora caninum was evaluated in dairy cattle of the Southwestern region of Mato Grosso State, Brazil, in addition to serum samples obtained from dogs and humans living in the farms. A total of 1036 serum samples were analyzed, from which 932 were from dairy bovine females, 37 from dogs and 67 from humans, from 24 farms and examined by the indirect fluorescent antibody test (IFAT). Reactive human scrum samples were retested by Western-blotting to confirm the results. Antibodies to N. caninum were found in 499 cattle sera (53.5 %), with at least one positive in each farm, 25 dog sera (67.6 %) and seven human sera (10.5 %). There was no significant difference in the number of positive cattle sera according to age group. The results indicate a wide dissemination of N caninum in the studied region.

Voltammetric immunosensor for the diagnosis of celiac disease based on the quantification of anti-gliadin antibodies

Antibodies against gliadin are used to detect celiac disease (CD) in patients. An electrochemical immunosensor for the voltammetric detection of human anti-gliadin antibodies (AGA) IgA and AGA IgG in real serum samples is proposed. The transducer surface consists of screen-printed carbon electrodes modified with a carbon nanotube/gold nanoparticle hybrid system, which provides a very useful surface for the amplification of the immunological interactions. The immunosensing strategy is based on the immobilization of gliadin, the antigen for the autoantibodies of interest, onto the nanostructured surface. The antigen–antibody interaction is recorded using alkaline phosphatase labeled anti-human antibodies and a mixture of 3-indoxyl phosphate with silver ions (3-IP/Ag+) was used as the substrate. The analytical signal is based on the anodic redissolution of the enzymatically generated silver by cyclic voltammetry. The electrochemical behavior of this immunosensor was carefully evaluated assessing aspects as sensitivity, non-specific binding and matrix effects, and repeatability and reproducibility. The results were supported with a commercial ELISA test.

Prevalence of anti-p: Falciparum sporozoite antibodies in adults in the amapa region of Brazil

17 of 20 adult sera from the Amapa region of Brazil were active in the inhibition of P. falciparum sporozoite invasion (ISI) assay which has been correlated with protective antibodies. In contrast 11 sera were positive in IFA tests and 6 were positive in CSP tests. These results suggest that the ISI assay will be useful for evaluating naturally acquired protective anti-sporozoite antibodies in endemic areas, particularly during vaccine efficacy studies using sporozoite-based vaccines.

Enzyme linked immunosorbent assay: determination of anti-adenovirus antibodies in an infant population

In order to define an accurate assay for anti-adenovirus antibody detection, a recently developed ELISA was compared with IFA and CF. On 58 sera, the ELISA was more sensitive than both CF and IFA, which showed relative sensitivities of 63% and 94%, respectively. It was not possible to determine the exact specificity of the tests because of the lack of a gold standard. Furthermore, the ELISA was used to define the prevalence of adenovirus antibodies in 116 infants between 1 and 24 months old (mean 7.28). The data showed that maternal antibodies waned by the age of 5 to 6 months and that more than 80% of the children had been infected by adenoviruses by the age of 10 months.

PRESENCE OF ANTI-TOXOCARA ANTIBODIES IN CHILDREN SELECTED AT HOSPITAL UNIVERSITÁRIO, CAMPO GRANDE, MS, BRAZIL

Visceral Larva Migrans syndrome (VLM) results from the presence or migration of helminth larvae in humans, who nonetheless only play the role of paratenic hosts in the helminths' life cycle. In humans, VLM can be caused by larvae of various nematode species, chiefly those of the ascarid Toxocara canis, which can then be found at a variety of body sites, such as the liver, lungs, heart, and brain. Clinical and pathological manifestations depend primarily on larvae number and location, infection duration, reinfection occurrence, and host's immunological condition. Signs and symptoms may range from asymptomatic infection to severe disease. In humans, infection is acquired through ingestion of T. canis eggs present in soil, containing larvae in the infective stage7, 8, 9. Indeed, eggs of Toxocara sp. have been found in sandboxes in several public places in the city of Campo Grande, Mato Grosso do Sul state2. This study was carried out to detect the presence of anti-Toxocara antibodies in children attending the Pediatrics division of Hospital Universitário of Universidade Federal de Mato Grosso do Sul at Campo Grande, Brazil. Over the years 1992-94, 454 serum samples, obtained from children of 5.25 ± 3.28 years of mean age and selected at that hospital on the basis of eosinophil count greater than or equal to 1000/mm3 of blood, were tested for the presence of antibodies by means of the ELISA technique employing Toxocara canis larvae excretory-secretory antigens5. A high prevalence rate for toxocariasis (35.55%) was found, which was observed to be associated with eosinophil levels lower than those usually reported in literature. Furthermore, a higher frequency of positive serology among boys was also observed (13 cases in contrast to only 3 among girls), a result also reported by other authors

PREVALENCE OF ANTI- Toxocara ANTIBODIES IN A RANDOM SAMPLE OF INPATIENTS AT A CHILDREN'S HOSPITAL IN VITÓRIA, ESPÍRITO SANTO, BRAZIL

In the streets of Vitória, in the State of Espírito Santo, Brazil, are large number of stray dogs, many of which are infected with Toxocara canis, suggesting a high risk for human infection. In order to investigate the prevalence of Toxocara infection in children in Espírito Santo we studied the prevalence of anti-Toxocara antibodies in 100 random inpatients over one year of age, at the Children's Hospital N.S. da Glória, the reference children's hospital for the State.All the sera were collected during the period between October 1996 and January 1997. The mean age was 6.6±4.1 yrs. (1 to 14 yrs., median 6yrs.) and there were patients from all of the different wards of the hospital. Sixty-eigth patients came from the metropolitan area of Vitória and the other 32 from 17 other municipalities. The anti-Toxocara antibodies were investigated by ELISA-IgG using a secretory-excretory antigen obtained from second stage larvae. All sera were adsorbed with Ascaris suum antigen before the test. Thirty-nine sera (39%) were positive, predominantly from boys, but the gender difference was not statistically significant (boys:25/56 or 44.6%; girls:14/44 or 31.8%; p=0.311). The prevalence of positive sera was higher, but not statistically significant, in children from the urban periphery of metropolitan Vitória (formed by the cities of Vitória, Cariacica, Vila Velha, Serra and Viana) than in children from 17 other municipalities (44.1% and 28.1% respectively, p=0.190). Although the samples studied do not represent all children living in the State of Espírito Santo, since the Children's Hospital N.S. da Glória admits only patients from the state health system, it is probable that these results indicate a high frequency of Toxocara infection in children living in Espírito Santo. Further studies of population samples are necessary to ascertain the prevalence of Toxocara infection in our country.

Production of monoclonal antibodies anti-Taenia crassiceps cysticerci with cross-reactivity with Taenia solium antigens

We describe the production of the potential monoclonal antibodies (MoAbs) using BALB/c mice immunized with vesicular fluid (VF)-Tcra (T. crassiceps) antigen. Immune sera presented anti-VF-Tcra (<20kD) IgG and IgM antibodies with cross-reactivity with T. solium (Tso) antigen (8-12, 14, and 18 kD). After cell fusion, we selected 33 anti-Tcra and anti-Tso reactive IgM-clones and 53 anti-Tcra specific IgG-clones, 5 of them also recognizing Tso antigens. Two clones identified the 8-14 and 18kD peptides of VF-Tcra.