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Article ; Online: Understanding hypergammaglobulinemia in experimental or natural visceral leishmaniasis.

de Carvalho, Camila Aparecida / Hiramoto, Roberto Mitsuyoshi / Meireles, Luciana Regina / de Andrade, Heitor Franco

2024 Volume 46, Issue 1, Page(s) e13021

Abstract: Nonspecific hypergammaglobulinemia (HGG) occurs in symptomatic human visceral leishmaniasis (VL) caused by L. L. infantum. This study assessed this finding in experimental infection in hamsters and natural infection in dogs. The serum concentration of ... ...

Abstract	Nonspecific hypergammaglobulinemia (HGG) occurs in symptomatic human visceral leishmaniasis (VL) caused by L. L. infantum. This study assessed this finding in experimental infection in hamsters and natural infection in dogs. The serum concentration of proteins, albumin and globulins was determined through the biuret and bromocresol green reaction, where the HGG was better expressed through the albumin/globulin (A/G) ratio. HGG was associated with a higher concentration of specific anti-glycan antibodies (BSA-G)/promastigote soluble extract (PSE) and the presence of circulating immune complexes (IC) by dissociative enzyme-linked immunoassay (ELISA). The study found monovalent IC in 37.9% (PSE) and 50% (BSA-G) of sera from infected hamsters, with increased frequency as the disease progressed. HGG was found in >60% of the samples in dogs with VL, associated with higher levels of specific immunoglobulin (Ig)A and IgM, but not IgG, determined using the PSE and BSA-G ELISA. HGG was associated with the presence of monovalent IC in 58.9% (PSE) and 63.4% (BSA-G) positive dog samples. HGG may result not only from the nonspecific activation of B cells, with greater production of specific and nonspecific antibodies, but also due to lower IgG excretion due to the presence of soluble monovalent IC. HGG correlates to the progression of VL and may be a marker for manifested disease.
MeSH term(s)	Cricetinae ; Humans ; Animals ; Dogs ; Leishmaniasis, Visceral ; Hypergammaglobulinemia ; Enzyme-Linked Immunosorbent Assay ; Antibodies, Protozoan ; Dog Diseases ; Leishmania infantum ; Antigen-Antibody Complex ; Albumins
Chemical Substances	Antibodies, Protozoan ; Antigen-Antibody Complex ; Albumins
Language	English
Publishing date	2024-01-26
Publishing country	England
Document type	Journal Article
ZDB-ID	424444-8
ISSN	1365-3024 ; 0141-9838
ISSN (online)	1365-3024
ISSN	0141-9838
DOI	10.1111/pim.13021
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Article ; Online: Evaluation of aversive behavior in Rattus norvegicus experimentally infected by two distinct strains of Toxoplasma gondii (ME49 and VEG): study of epigenetic markers.

Santos, Sergio Vieira Dos / Fortuna, Giulio de Los Santos / Barbosa, Lariane Monteiro / Meireles, Luciana Regina / Tiago, Érico Silva / Chieffi, Pedro Paulo

Revista da Sociedade Brasileira de Medicina Tropical

2022 Volume 55, Page(s) e01222022

Abstract: Background: Behavioral changes in Rattus norvegicus infected with two strains of Toxoplasma gondii (ME49 and VEG) were investigated.: Methods: Rats were evaluated for motor activity and aversion or attraction to cat urine 60 days after infection. ... ...

Abstract	Background: Behavioral changes in Rattus norvegicus infected with two strains of Toxoplasma gondii (ME49 and VEG) were investigated. Methods: Rats were evaluated for motor activity and aversion or attraction to cat urine 60 days after infection. After euthanasia, arginine-vasopressin gene methylation in the central nervous system was evaluated. Results: A significant difference was observed in the methylation of the arginine-vasopressin promoter gene between rats infected with the ME49 and VEG strains. Conclusions: Although differences were not observed in many parameters, significant differences were observed in the methylation of the arginine-vasopressin promoter gene in rats infected with the two studied strains.
MeSH term(s)	Rats ; Animals ; Toxoplasma/genetics ; Behavior, Animal/physiology ; Epigenesis, Genetic ; Vasopressins/genetics ; Arginine/genetics ; Toxoplasmosis, Animal
Chemical Substances	Vasopressins (11000-17-2) ; Arginine (94ZLA3W45F)
Language	English
Publishing date	2022-10-24
Publishing country	Brazil
Document type	Journal Article
ZDB-ID	1038126-0
ISSN	1678-9849 ; 0037-8682
ISSN (online)	1678-9849
ISSN	0037-8682
DOI	10.1590/0037-8682-0122-2022
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Article ; Online: Serum antibodies blocked by glycan antigens in canine visceral leishmaniasis serology are mostly IgA immune complexes.

Aparecida de Carvalho, Camila / Mitsuyoshi Hiramoto, Roberto / Regina Meireles, Luciana / Franco de Andrade Júnior, Heitor

Parasitology

2021 Volume 148, Issue 12, Page(s) 1509–1515

Abstract: Immune complexes (ICs) are found in canine visceral leishmaniasis (CVL) and interfere with the serum detection of antibodies. Dissociation of these monovalent complexes by dissociative enzyme-linked immunosorbent assay (ELISA) removes false-negative ... ...

Abstract	Immune complexes (ICs) are found in canine visceral leishmaniasis (CVL) and interfere with the serum detection of antibodies. Dissociation of these monovalent complexes by dissociative enzyme-linked immunosorbent assay (ELISA) removes false-negative results and allows some characterization of antibodies and antigens. We studied the serology of dogs with suspected CVL in an endemic area, testing two Leishmania (Leishmania) [L. (L.)] <full form>infantum antigens. We analysed the presence of immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin M (IgM) antibodies specific to promastigote soluble extract (PSE) and low-molecular weight glycans (glycan–bovine serum albumin (BSA) complex – GBC) by conventional and dissociative ELISA. Our results showed a significant fraction of IgA ICs (46.5% for PSE and 47.6% for GBC), followed by IgG ICs (10% for PSE and 23.5% for GBC). IgM ICs were more frequent for PSE (22.7%). Hypergammaglobulinaemia in CVL would be related to the presence of IgA and IgG ICs, resulting in deficient elimination of these antibodies. Our data confirmed the presence of ICs that can generate false-negative results in conventional serology. The production of IgA antibodies and the high frequency of blockade by glycan antigens suggest the active participation of this immunoglobulin and its ICs in the immunopathology of CVL, indicating a new path for further research.
MeSH term(s)	Animals ; Antigen-Antibody Complex ; Dog Diseases/diagnosis ; Dogs ; Immunoglobulin A ; Leishmaniasis, Visceral/diagnosis ; Leishmaniasis, Visceral/veterinary ; Polysaccharides
Chemical Substances	Antigen-Antibody Complex ; Immunoglobulin A ; Polysaccharides
Language	English
Publishing date	2021-07-05
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	207627-5
ISSN	1469-8161 ; 0031-1820
ISSN (online)	1469-8161
ISSN	0031-1820
DOI	10.1017/S0031182021001189
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Article ; Online: Toxoplasma gondii isolated from a Brazilian patient with rare pulmonary toxoplasmosis has a novel genotype and is closely related to Amazonian isolates.

Pena, Hilda Fátima Jesus / Ferreira, Marina Neves / Gennari, Solange Maria / de Andrade, Heitor Franco / Meireles, Luciana Regina / Galisteo, Andrés Jimenez

Parasitology research

2021 Volume 120, Issue 3, Page(s) 1109–1113

Abstract: Pulmonary toxoplasmosis is rare in immunocompetent patients. Herein, a Toxoplasma gondii strain isolated in Brazil from an immunocompetent patient who had severe pulmonary involvement was biologically and molecularly characterized for the first time. The ...

Abstract	Pulmonary toxoplasmosis is rare in immunocompetent patients. Herein, a Toxoplasma gondii strain isolated in Brazil from an immunocompetent patient who had severe pulmonary involvement was biologically and molecularly characterized for the first time. The TgHumIMTBr1 isolate was bioassayed in mice showing a virulent phenotype. Restriction fragment length polymorphism (RFLP) genotyping using 11 markers [SAG1, SAG2 (5´3´SAG2 and alt. SAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico and CS3] revealed a new non-archetypal genotype assigned as #312. Genotyping using ROP18/ROP5 markers exhibited the virulent combination of alleles 4 and 1. Microsatellite analysis using 15 markers (TUB2, W35, TgM-A, B18, B17, M33, IV.1, X1.1, N60, N82, AA, N61, N83, M48 and M102) revealed an atypical genotype with three unique alleles and a rare combination of alleles 246 (W35) and 203 (TgM-A) that is typical of the Amazon region. Non-archetypal genotypes with unique alleles may function in the occurrence of severe toxoplasmosis in immunocompetent patients in Brazil. Attempts to isolate or molecularly detect T. gondii for further genotyping studies would contribute to the understanding of causes related to the severity of toxoplasmosis in immunocompetent patients.
MeSH term(s)	Adult ; Alleles ; Animals ; Brazil ; Genetic Variation ; Genotype ; Humans ; Immunocompetence ; Lung Diseases, Parasitic/parasitology ; Male ; Mice ; Phylogeny ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Toxoplasma/classification ; Toxoplasma/genetics ; Toxoplasma/isolation & purification ; Toxoplasmosis/parasitology
Language	English
Publishing date	2021-01-09
Publishing country	Germany
Document type	Case Reports ; Journal Article
ZDB-ID	284966-5
ISSN	1432-1955 ; 0932-0113 ; 0044-3255
ISSN (online)	1432-1955
ISSN	0932-0113 ; 0044-3255
DOI	10.1007/s00436-020-07008-4
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Uh II Zs.124: Show issues

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Article ; Online: Early high avidity specific IgG production in experimental hamster visceral leishmaniasis.

de Carvalho, Camila Aparecida / Ferrão, Thiago Fidelis / Cavalcante, Fernanda Siqueira / de Freitas, Flavia Regina Novais / Meireles, Luciana Regina / de Andrade Júnior, Heitor Franco

Parasitology research

2020 Volume 119, Issue 11, Page(s) 3881–3885

Abstract: Visceral leishmaniasis (VL) by Leishmania (Leishmania) infantum is epidemic in Brazil. Hypergammaglobulinemia appears early in patients with VL and is ineffective. Usually, high-affinity IgG B cells are selected during most infections, a critical step ... ...

Abstract	Visceral leishmaniasis (VL) by Leishmania (Leishmania) infantum is epidemic in Brazil. Hypergammaglobulinemia appears early in patients with VL and is ineffective. Usually, high-affinity IgG B cells are selected during most infections, a critical step for an effective humoral response. The avidity of IgG antibodies in VL is unexplored due to the absence of temporal parameters in most patients, associated to low clinical significance. Experimental infection models overcome this fact, allowing the monitoring of the disease temporal evolution. In this study, the avidity of IgG antibodies was evaluated in experimental models, in infection in hamsters, and in immunization in rabbits. Specific IgG antibodies were detected by ELISA, using chaotropic solution to determine avidity, as reported for viral infections. The levels of IgG antibodies correlated with the progression of experimental infection in hamsters or antigenic stimulation in immunized rabbits. However, IgG avidity was high early in infected animals, even in early periods (> 80%), while in immunized rabbits, they had early antibodies of low avidity with progressive maturation, similar as other infections. These data suggest that the affinity maturation of the avidity of anti-Leishmania IgG antibodies promoted at an early stage, influencing the appropriate interaction between antigens and affecting the disease progression. This fact could be associated to monovalent immune complexes, as reported in human and experimental VL. This scenario may be related to an independent process of immune cell activation by the parasite but absent in antigen preparation used as immunogens.
MeSH term(s)	Animals ; Antibodies, Protozoan/blood ; Antibodies, Protozoan/immunology ; Antigen-Antibody Complex/immunology ; Brazil ; Cricetinae ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Immunoglobulin G/blood ; Immunoglobulin G/immunology ; Leishmania infantum/immunology ; Leishmaniasis, Visceral/immunology ; Male ; Rabbits ; Vaccination
Chemical Substances	Antibodies, Protozoan ; Antigen-Antibody Complex ; Immunoglobulin G
Language	English
Publishing date	2020-08-14
Publishing country	Germany
Document type	Journal Article
ZDB-ID	284966-5
ISSN	1432-1955 ; 0932-0113 ; 0044-3255
ISSN (online)	1432-1955
ISSN	0932-0113 ; 0044-3255
DOI	10.1007/s00436-020-06851-9
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Article: Serum antibodies blocked by glycan antigens in canine visceral leishmaniasis serology are mostly IgA immune complexes

Aparecida de Carvalho, Camila / Mitsuyoshi Hiramoto, Roberto / Regina Meireles, Luciana / Franco de Andrade Júnior, Heitor

Parasitology. 2021 Oct., v. 148, no. 12

2021

Abstract	Immune complexes (ICs) are found in canine visceral leishmaniasis (CVL) and interfere with the serum detection of antibodies. Dissociation of these monovalent complexes by dissociative enzyme-linked immunosorbent assay (ELISA) removes false-negative results and allows some characterization of antibodies and antigens. We studied the serology of dogs with suspected CVL in an endemic area, testing two Leishmania (Leishmania) [L. (L.)] <full form>infantum antigens. We analysed the presence of immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin M (IgM) antibodies specific to promastigote soluble extract (PSE) and low-molecular weight glycans (glycan–bovine serum albumin (BSA) complex – GBC) by conventional and dissociative ELISA. Our results showed a significant fraction of IgA ICs (46.5% for PSE and 47.6% for GBC), followed by IgG ICs (10% for PSE and 23.5% for GBC). IgM ICs were more frequent for PSE (22.7%). Hypergammaglobulinaemia in CVL would be related to the presence of IgA and IgG ICs, resulting in deficient elimination of these antibodies. Our data confirmed the presence of ICs that can generate false-negative results in conventional serology. The production of IgA antibodies and the high frequency of blockade by glycan antigens suggest the active participation of this immunoglobulin and its ICs in the immunopathology of CVL, indicating a new path for further research.
Keywords	Leishmania ; blood serum ; dogs ; enzyme-linked immunosorbent assay ; immunoglobulin A ; immunoglobulin G ; immunopathology ; parasitology ; polysaccharides ; serology ; visceral leishmaniasis
Language	English
Dates of publication	2021-10
Size	p. 1509-1515.
Publishing place	Cambridge University Press
Document type	Article
ZDB-ID	207627-5
ISSN	1469-8161 ; 0031-1820
ISSN (online)	1469-8161
ISSN	0031-1820
DOI	10.1017/S0031182021001189
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Isolation and characterization of Toxoplasma gondii isolates from human congenital toxoplasmosis cases reveal a new virulent genotype in São Paulo, Brazil.

Meireles, Luciana Regina / Bezerra, Elizama Carneiro Machado / Andrade, Joelma Queiroz / Cassiano, Larissa Aparecida / Pena, Hilda Fátima Jesus / Alves, Bruna Farias / Francisco, Rossana Pulcineli Vieira / de Andrade, Heitor Franco

Parasitology research

2022 Volume 121, Issue 11, Page(s) 3223–3228

Abstract: Toxoplasma gondii causes severe disease in congenitally infected fetuses. The severity of fetal infection is related to the gestational stage at the time of maternal infection, parasite burden, and genotypic characteristics. South America has a high ... ...

Abstract	Toxoplasma gondii causes severe disease in congenitally infected fetuses. The severity of fetal infection is related to the gestational stage at the time of maternal infection, parasite burden, and genotypic characteristics. South America has a high incidence of congenital toxoplasmosis and has the highest genotypic diversity of the parasite. In Brazil, clinical toxoplasmosis in children is notorious, however there are very limited data regarding the strains recovered from congenital infections. In this study, T. gondii strains from two cases of severe congenital toxoplasmosis from the São Paulo metropolitan area were isolated (TgHumIMTBr2 and TgHumIMTBr3) and biologically and molecularly characterized using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and microsatellite analysis, revealing a new non-archetypal virulent genotype designated as #318. The other isolate, genotype #175, has already been described in domestic and wild animals in Brazil, but is now associated with acute toxoplasmosis in humans. These data reinforce the role of non-archetypal T. gondii genotypes in the severity of human congenital toxoplasmosis, highlighting the importance of studies focused on parasite isolation and genotyping for a better understanding of the virulence of isolates from human toxoplasmosis and contributing to the knowledge of the diversity of T. gondii in Brazil.
MeSH term(s)	Brazil/epidemiology ; Child ; Genetic Variation ; Genotype ; Humans ; Polymorphism, Restriction Fragment Length ; Toxoplasma/genetics ; Toxoplasmosis, Congenital/epidemiology ; Toxoplasmosis, Congenital/parasitology
Language	English
Publishing date	2022-09-02
Publishing country	Germany
Document type	Journal Article
ZDB-ID	284966-5
ISSN	1432-1955 ; 0932-0113 ; 0044-3255
ISSN (online)	1432-1955
ISSN	0932-0113 ; 0044-3255
DOI	10.1007/s00436-022-07643-z
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Uh II Zs.124: Show issues

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Article ; Online: Saliva collection and detection of anti- T. gondii antibodies of low-income school-age children as a learning strategy on hygiene, prevention and transmission of toxoplasmosis.

Macre, Miriam de Souza / Meireles, Luciana Regina / Sampaio, Barbara Fialho Carvalho / Andrade Júnior, Heitor Franco de

Revista do Instituto de Medicina Tropical de Sao Paulo

2019 Volume 61, Page(s) e48

Abstract: School-age children are a social group in which blood collection for laboratory testing can be perceived as an invasive procedure, with low acceptance and tolerance of stakeholders. This problem could be circumvented by replacing serum samples with ... ...

Abstract	School-age children are a social group in which blood collection for laboratory testing can be perceived as an invasive procedure, with low acceptance and tolerance of stakeholders. This problem could be circumvented by replacing serum samples with saliva. For this purpose, and to make the collection of saliva samples playful and instructive for children, educational activities on hygiene and toxoplasmosis transmission and prevention were performed using toys and audiovisual tools. The target audience consisted of 7-10 year-old children from low-income families who attended public schools in the city of São Paulo. Saliva samples were used in a previously described in-house Enzyme-Linked Immunosorbent Assays (ELISA) to detect anti- Toxoplasma gondii IgG antibodies and establish the immunological status of each of the participants. One year later, children's memory and fixation of concepts regarding hygiene habits, as well as transmission and prevention of toxoplasmosis were tested in the same schools, by means of a questionnaire application, using students who did not participate in the first intervention as controls. The prevalence of positive anti- T. gondii IgG among students was 50% (82/164). One year later, 45 children had more knowledge on toxoplasmosis (28/45 vs 29/147) and they drew the cat's involvement in the transmission of toxoplasmosis more often than controls (28/45 vs 29/147). Sorted according to the presence of specific IgG in saliva, recovered positive students presented worse memory of the above cited knowledge as did saliva-negative IgG students, but both groups had isolated higher frequency of fixed knowledge than non-intervened students. Our data show that there is a high prevalence of T. gondii infection in school-children from low-income areas; saliva is an alternative to blood for anti- T. gondii IgG detection; and a one-day educational intervention in school-children was effective in promoting knowledge fixation on hygiene and toxoplasmosis transmission and prevention after one year.
MeSH term(s)	Antibodies, Protozoan/analysis ; Child ; Enzyme-Linked Immunosorbent Assay ; Female ; Health Education/methods ; Health Knowledge, Attitudes, Practice ; Humans ; Hygiene ; Immunoglobulin G/analysis ; Male ; Poverty ; Saliva/parasitology ; Surveys and Questionnaires ; Toxoplasma/immunology
Chemical Substances	Antibodies, Protozoan ; Immunoglobulin G
Language	English
Publishing date	2019-09-12
Publishing country	Brazil
Document type	Journal Article
ZDB-ID	128928-7
ISSN	1678-9946 ; 0036-4665
ISSN (online)	1678-9946
ISSN	0036-4665
DOI	10.1590/S1678-9946201961048
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Article ; Online: Measles, rubella, mumps and Toxoplasma gondii antibodies in saliva of vaccinated students of schools and universities in São Paulo City, Brazil.

Sampaio, Barbara Carvalho Fialho / Rodrigues, Jaqueline Polizeli / Meireles, Luciana Regina / Andrade Junior, Heitor Franco de

The Brazilian journal of infectious diseases : an official publication of the Brazilian Society of Infectious Diseases

2019 Volume 24, Issue 1, Page(s) 51–57

Abstract: Introduction: Vaccines are well-established public health interventions with major impact on the prevalence of infectious diseases, but outbreaks are occurring frequently due to primary and secondary failures, despite high coverage. Surveillance of ... ...

Abstract	Introduction: Vaccines are well-established public health interventions with major impact on the prevalence of infectious diseases, but outbreaks are occurring frequently due to primary and secondary failures, despite high coverage. Surveillance of efficacy and duration of induced immunity is a difficult task as it requires invasive blood sampling in children and teenagers. Saliva can be an acceptable alternative source of IgG to assess vaccine efficacy and toxoplasmosis incidence. We investigated IgG response for measles, mumps, rubella, and T. gondii in saliva samples of vaccinated young people. Methods: Saliva was collected from 249 public schools students from São Paulo, Brazil, aged 7 to 13 years old, during an interactive exhibition on hygiene. We used S. aureus protein A solid phase capture assay for IgG reactive to biotinylated purified proteins. Paired saliva and serum (47) were tested from young adults with serum evidence of T. gondii infection and from negative children less than 12 month old for standardization. Reproducibility was greater than 98% and sensitivity and specificity of the saliva assays were greater than 95%, as well as the concordance of paired saliva and serum samples. Results: Saliva from high school students showed a prevalence of 8.5% (95% CI: 5.0-11.9%) for anti T. gondii IgG; 96.8% (94.6-99%) of anti-measles IgG; 59.1% (53-65%) of anti-rubella IgG, and 57.5% (51.3-63.6%) of anti-mumps IgG. Discussion: The prevalence of antibodies against mumps and rubella after 6-8 years of vaccination was lower than against measles among students. The findings of this study demonstrate the feasibility of saliva sampling for follow-up of vaccine immune status in teenagers. This useful approach allows for IgG detection for vaccine control or epidemiological studies.
MeSH term(s)	Adolescent ; Antibodies, Protozoan/analysis ; Antibodies, Viral/analysis ; Brazil ; Child ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Immunoenzyme Techniques ; Immunoglobulin G/analysis ; Immunoglobulin G/immunology ; Male ; Measles/immunology ; Measles/prevention & control ; Measles-Mumps-Rubella Vaccine/immunology ; Mumps/immunology ; Mumps/prevention & control ; ROC Curve ; Reference Values ; Rubella/immunology ; Rubella/prevention & control ; Saliva/immunology ; Students/statistics & numerical data ; Toxoplasmosis/immunology ; Toxoplasmosis/prevention & control
Chemical Substances	Antibodies, Protozoan ; Antibodies, Viral ; Immunoglobulin G ; Measles-Mumps-Rubella Vaccine
Language	English
Publishing date	2019-12-19
Publishing country	Brazil
Document type	Journal Article
ZDB-ID	2041400-6
ISSN	1678-4391 ; 1413-8670
ISSN (online)	1678-4391
ISSN	1413-8670
DOI	10.1016/j.bjid.2019.11.005
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Article ; Online: High level SARS-CoV-2 nucleocapsid refolding using mild condition for inclusion bodies solubilization: Application of high pressure at pH 9.0.

Chura-Chambi, Rosa Maria / Prieto-da-Silva, Alvaro Rossan de Brandão / Di Lela, Matheus Martins / Oliveira, João Ezequiel / Abreu, Patricia Estima Antonia / Meireles, Luciana Regina / de Andrade Junior, Heitor Franco / Morganti, Ligia

PloS one

2022 Volume 17, Issue 2, Page(s) e0262591

Abstract: SARS-CoV-2 Nucleocapsid (N) is the most abundant viral protein expressed in host samples and is an important antigen for diagnosis. N is a 45 kDa protein that does not present disulfide bonds. Intending to avoid non-specific binding of SARS-CoV-2 N to ... ...

Abstract	SARS-CoV-2 Nucleocapsid (N) is the most abundant viral protein expressed in host samples and is an important antigen for diagnosis. N is a 45 kDa protein that does not present disulfide bonds. Intending to avoid non-specific binding of SARS-CoV-2 N to antibodies from patients who previously had different coronaviruses, a 35 kDa fragment of N was expressed without a conserved motif in E. coli as inclusion bodies (N122-419-IB). Culture media and IB washing conditions were chosen to obtain N122-419-IB with high yield (370 mg/L bacterial culture) and protein purity (90%). High pressure solubilizes protein aggregates by weakening hydrophobic and ionic interactions and alkaline pH promotes solubilization by electrostatic repulsion. The association of pH 9.0 and 2.4 kbar promoted efficient solubilization of N122-419-IB without loss of native-like tertiary structure that N presents in IB. N122-419 was refolded with a yield of 85% (326 mg/L culture) and 95% purity. The refolding process takes only 2 hours and the protein is ready for use after pH adjustment, avoiding the necessity of dialysis or purification. Antibody binding of COVID-19-positive patients sera to N122-419 was confirmed by Western blotting. ELISA using N122-419 is effective in distinguishing between sera presenting antibodies against SARS-CoV-2 from those who do not. To the best of our knowledge, the proposed condition for IB solubilization is one of the mildest described. It is possible that the refolding process can be extended to a wide range of proteins with high yields and purity, even those that are sensible to very alkaline pH.
MeSH term(s)	Antibodies, Viral/blood ; Antibodies, Viral/immunology ; Antigens, Viral/chemistry ; Antigens, Viral/immunology ; COVID-19/blood ; COVID-19/diagnosis ; COVID-19/virology ; Coronavirus Nucleocapsid Proteins/chemistry ; Coronavirus Nucleocapsid Proteins/immunology ; Enzyme-Linked Immunosorbent Assay/methods ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Humans ; Hydrogen-Ion Concentration ; Hydrostatic Pressure ; Immunoglobulin G/blood ; Immunoglobulin G/immunology ; Inclusion Bodies/chemistry ; Phosphoproteins/chemistry ; Phosphoproteins/immunology ; Protein Refolding ; Protein Structure, Tertiary ; Recombinant Proteins/chemistry ; Recombinant Proteins/immunology ; SARS-CoV-2/immunology ; Solubility
Chemical Substances	Antibodies, Viral ; Antigens, Viral ; Coronavirus Nucleocapsid Proteins ; Immunoglobulin G ; Phosphoproteins ; Recombinant Proteins ; nucleocapsid phosphoprotein, SARS-CoV-2
Language	English
Publishing date	2022-02-03
Publishing country	United States
Document type	Journal Article
ZDB-ID	2267670-3
ISSN	1932-6203 ; 1932-6203
ISSN (online)	1932-6203
ISSN	1932-6203
DOI	10.1371/journal.pone.0262591
Database	MEDical Literature Analysis and Retrieval System OnLINE

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