Development and Validation of a Novel Diagnostic Test for Human Brucellosis Using a Glyco-engineered Antigen Coupled to Magnetic Beads.

Brucellosis is a highly contagious zoonosis and still a major human health problem in endemic areas of the world. Although several diagnostic tools are available, most of them are difficult to implement especially in developing countries where complex health facilities are limited. Taking advantage of the identical structure and composition of the Brucella spp. and Yersinia enterocolitica O:9 O-polysaccharide, we explored the application of a recombinant Y. enterocolitica O:9-polysaccharide-protein conjugate (OAg-AcrA) as a novel antigen for diagnosis of human brucellosis. We have developed and validated an indirect immunoassay using OAg-AcrA coupled to magnetic beads. OAg-AcrA was produced and purified with high yields in Y. enterocolitica O:9 cells co-expressing the oligosaccharyltransferase PglB and the protein acceptor AcrA of Campylobacter jejuni without the need for culturing Brucella. Expression of PglB and AcrA in Y. enterocolitica resulted in the transfer of the host O-polysaccharide from its lipid carrier to AcrA. To validate the assay and determine the cutoff values a receiver-operating characteristic analysis was performed using a panel of characterized serum samples obtained from healthy individuals and patients of different clinical groups. Our results indicate that, using this assay, it is possible to detect infection caused by the three main human brucellosis agents (B. abortus, B. melitensis and B. suis) and select different cutoff points to adjust sensitivity and specificity levels as needed. A cutoff value of 13.20% gave a sensitivity of 100% and a specificity of 98.57%, and a cutoff value of 16.15% resulted in a test sensitivity and specificity of 93.48% and 100%, respectively. The high diagnostic accuracy, low cost, reduced assay time and simplicity of this new glycoconjugate-magnetic beads assay makes it an attractive diagnostic tool for using not only in clinics and brucellosis reference laboratories but also in locations with limited laboratory infrastructure and/or minimally trained community health workers.Fil: Ciocchini, Andres Eduardo. Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico Chascomús (San Martin); Argentina;Fil: Rey Serantes, Diego A.. Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico Chascomús (San Martin); Argentina;Fil: Melli, Luciano Jorge. Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico Chascomús (San Martin); Argentina;Fil: Iwashkiw, Jeremy A.. University of Alberta . Department of Biological Sciences . Alberta Glycomics Centre; Estados Unidos de América;Fil: Deodato, Bettina. Hospital Múñiz. Unidad de Enfermedades Infecciosas; Argentina;Fil: Wallach, Jorge. Hospital Múñiz. Unidad de Enfermedades Infecciosas; Argentina;Fil: Feldman, Mario F. University of Alberta . Department of Biological Sciences . Alberta Glycomics Centre; Estados Unidos de América;Fil: Ugalde, Juan E. Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico Chascomús (San Martin); Argentina;Fil: Comerci, Diego J. Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico Chascomús (San Martin); Argentina

Diminished production of T helper 1 cytokines correlates with T cell unresponsiveness to Brucella cytoplasmic proteins in chronic human brucellosis

This study evaluated the cellular immune response against Brucella species cytoplasmic protein (CP) in peripheral blood mononuclear cells (PBMC) of 25 patients with brucellosis. In vitro proliferation and cytokine gene expression and production were investigated. PBMC from 14 patients proliferated in response to CP (responder patients [RPs]) and cells from 11 patients did not (nonresponder patients [NRPs]). CP-specific interleukin (IL)-2 and interferon-γ were significantly induced in PBMC from RPs, compared with cells from NRPs. No significant differences were found in the production of IL-10 between the 2 groups. CP did not induce IL-4 production. A close relationship was observed between the clinical status of the patients and the T cell response against CP. Patient with acute infections responded to CP and induced production of T helper 1 (Th1) cytokines, whereas chronically infected patients did not. Diminished production of Th1 cytokines may contribute to T cell unresponsiveness in chronic human brucellosis.Facultad de Ciencias Exacta

Human Infection with M- Strain of Brucella canis

The less mucoid strain of Brucella canis or M- strain is used for the serologic diagnosis of canine brucellosis. While this strain is avirulent in dogs, we report the case of clinical brucellosis that developed in a laboratory worker a few days after handling live M- cells for antigen production.Facultad de Ciencias Exacta

Human Infection with M- Strain of Brucella canis

The less mucoid strain of Brucella canis or M- strain is used for the serologic diagnosis of canine brucellosis. While this strain is avirulent in dogs, we report the case of clinical brucellosis that developed in a laboratory worker a few days after handling live M- cells for antigen production

Diminished production of T helper 1 cytokines correlates with T cell unresponsiveness to Brucella cytoplasmic proteins in chronic human brucellosis

This study evaluated the cellular immune response against Brucella species cytoplasmic protein (CP) in peripheral blood mononuclear cells (PBMC) of 25 patients with brucellosis. In vitro proliferation and cytokine gene expression and production were investigated. PBMC from 14 patients proliferated in response to CP (responder patients [RPs]) and cells from 11 patients did not (nonresponder patients [NRPs]). CP-specific interleukin (IL)-2 and interferon-γ were significantly induced in PBMC from RPs, compared with cells from NRPs. No significant differences were found in the production of IL-10 between the 2 groups. CP did not induce IL-4 production. A close relationship was observed between the clinical status of the patients and the T cell response against CP. Patient with acute infections responded to CP and induced production of T helper 1 (Th1) cytokines, whereas chronically infected patients did not. Diminished production of Th1 cytokines may contribute to T cell unresponsiveness in chronic human brucellosis.Facultad de Ciencias Exacta

MTADV 5-MER peptide suppresses chronic inflammations as well as autoimmune pathologies and unveils a new potential target-Serum Amyloid A.

Despite the existence of potent anti-inflammatory biological drugs e.g., anti-TNF and anti IL-6 receptor antibodies, for treating chronic inflammatory and autoimmune diseases, these are costly and not specific. Cheaper oral available drugs remain an unmet need. Expression of the acute phase protein Serum Amyloid A (SAA) is dependent on release of pro-inflammatory cytokines IL-1, IL-6 and TNF-α during inflammation. Conversely, SAA induces pro-inflammatory cytokine secretion, including Th17, leading to a pathogenic vicious cycle and chronic inflammation. 5- MER peptide (5-MP) MTADV (methionine-threonine-alanine-aspartic acid-valine), also called Amilo-5MER, was originally derived from a sequence of a pro-inflammatory CD44 variant isolated from synovial fluid of a Rheumatoid Arthritis (RA) patient. This human peptide displays an efficient anti-inflammatory effects to ameliorate pathology and clinical symptoms in mouse models of RA, Inflammatory Bowel Disease (IBD) and Multiple Sclerosis (MS). Bioinformatics and qRT-PCR revealed that 5-MP, administrated to encephalomyelytic mice, up-regulates genes contributing to chronic inflammation resistance. Mass spectrometry of proteins that were pulled down from an RA synovial cell extract with biotinylated 5-MP, showed that it binds SAA. 5-MP disrupted SAA assembly, which is correlated with its pro-inflammatory activity. The peptide MTADV (but not scrambled TMVAD) significantly inhibited the release of pro-inflammatory cytokines IL-6 and IL-1β from SAA-activated human fibroblasts, THP-1 monocytes and peripheral blood mononuclear cells. 5-MP suppresses the pro-inflammatory IL-6 release from SAA-activated cells, but not from non-activated cells. 5-MP could not display therapeutic activity in rats, which are SAA deficient, but does inhibit inflammations in animal models of IBD and MS, both are SAA-dependent, as shown by others in SAA knockout mice. In conclusion, 5-MP suppresses chronic inflammation in animal models of RA, IBD and MS, which are SAA-dependent, but not in animal models, which are SAA-independent

Dynamics of Adrenal Steroids Are Related to Variations in Th1 and Treg Populations during Mycobacterium tuberculosis Infection in HIV Positive Persons

Tuberculosis (TB) remains the most frequent cause of illness and death from an infectious agent, and its interaction with HIV has devastating effects. We determined plasma levels of dehydroepiandrosterone (DHEA), its circulating form DHEA-suphate (DHEA-s) and cortisol in different stages of M. tuberculosis infection, and explored their role on the Th1 and Treg populations during different scenarios of HIV-TB coinfection, including the immune reconstitution inflammatory syndrome (IRIS), a condition related to antiretroviral treatment. DHEA levels were diminished in HIV-TB and HIV-TB IRIS patients compared to healthy donors (HD), HIV+ individuals and HIV+ individuals with latent TB (HIV-LTB), whereas dehydroepiandrosterone sulfate (DHEA-s) levels were markedly diminished in HIV-TB IRIS individuals. HIV-TB and IRIS patients presented a cortisol/DHEA ratio significantly higher than HIV+, HIV-LTB and HD individuals. A positive correlation was observed between DHEA-s and CD4 count among HIV-TB individuals. Conversely, cortisol plasma level inversely correlated with CD4 count within HIV-TB individuals. M. tuberculosis-specific Th1 lymphocyte count was increased after culturing PBMC from HIV-TB individuals in presence of DHEA. We observed an inverse correlation between DHEA-s plasma level and Treg frequency in co-infected individuals, and CD4+FoxP3+ Treg frequency was increased in HIV-TB and IRIS patients compared to other groups. Strikingly, we observed a prominent CD4+CD25-FoxP3+ population across HIV-TB and HIV-TB IRIS patients, which frequency correlated with DHEA plasma level. Finally, DHEA treatment negatively regulated FoxP3 expression without altering Treg frequency in co-infected patients. These data suggest an enhancing role for DHEA in the immune response against M. tuberculosis during HIV-TB coinfection and IRIS

Limited diagnostic usefulness of antibodies to cytoplasmic proteins of Brucella in early-treated human brucellosis.

Antibodies to cytoplasmic proteins (CP) of Brucella have been shown to be useful for the diagnosis of human brucellosis; however, some early-diagnosed patients lack such an antibody response while having high titers of antibodies to lipopolysaccharide (LPS). To address which factors determine this serological discrepancy in the early stages of brucellosis we examined the antibody response to CP and LPS of 21 patients involved in an outbreak of B. melitensis infection who had a short duration of clinical illness at diagnosis (3–40 d). At diagnosis, antibodies to LPS (IgM and/or IgG) were found in all patients, while anti-CP antibodies were detected in 16 subjects (76%). At 6 weeks post-diagnosis IgG to CP (with or without IgM) had been detected in 13 patients and IgM alone had been found in 4; however, 4 other patients (19%) had no response to CP. No signi cant differences were found between these 3 groups in terms of age, gender, antimicrobial agents or factors that could hamper the immune response. Notably, however, the 4 non-responders and 3 of the 4 patients having only IgM to CP had started antibiotic therapy within 14 d post-symptoms, while treatment was started later in 9 of 13 patients who developed anti-CP IgG. In addition, maximum titers of IgG to CP tended to be lower in early-treated patients. These results suggest that very early antibiotic therapy hampers the antibody response to Brucella CP but has little impact on the anti-LPS response. Given the higher speci city of the former and the higher sensitivity of the latter, both reactivities should be measured in order to diagnose human brucellosis.Fil: Baldi, Pablo Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Giambartolomei, Guillermo Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; ArgentinaFil: Wallach, Jorge Carlos. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas "Dr. Francisco Javier Muñiz"; ArgentinaFil: Alejandro Velikovsky, Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Fossati, Carlos Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; Argentin

Antibody Reactivity to Omp31 from Brucella melitensis in Human and Animal Infections by Smooth and Rough Brucellae

Group 3 of outer membrane proteins (OMPs) of Brucella includes Omp25 and Omp31, which share 34% identity. Omp25 is highly conserved in Brucella species, and Omp31 is present in all Brucella species, except Brucella abortus. Antibodies to Brucella melitensis Omp31 have been sought only in infected sheep, and Western blotting of sera from infected sheep did not reveal anti-Omp31 reactivity. We obtained recombinant purified Omp31 (B. melitensis) and tested its recognition by sera from humans and animals suffering from brucellosis by an indirect enzyme-linked immunosorbent assay (ELISA). Serum samples from 74 patients, 57 sheep, and 47 dogs were analyzed; brucellosis was confirmed by bacteriological isolation in all ovine and canine cases and 31 human cases of brucellosis. Thirty-five patients (47%) were positive for antibodies to Omp31, including seven cases of Brucella suis infection, two cases of B. abortus infection, and three cases of B. melitensis infection. Of 39 sheep naturally infected with B. melitensis (biovars 1 and 3), 23 (59%) were positive for antibodies to Omp31. Anti-Omp31 antibodies were also detected in 12 of 18 rams (67%) in which Brucella ovis was isolated from semen. Antibodies to Omp31 were also found in 41 (87%) of the 47 dogs, including 13 with recent infection. These results suggest that an indirect ELISA using recombinant purified Omp31 from B. melitensis would be of limited value for the diagnosis of human and animal brucellosis. Nevertheless, the potential usefulness of this antigen in combination with other recombinant proteins from Brucella should not be dismissed.