RBD and Spike DNA-Based Immunization in Rabbits Elicited IgG Avidity Maturation and High Neutralizing Antibody Responses against SARS-CoV-2.

Neutralizing antibodies (nAbs) are a critical part of coronavirus disease 2019 (COVID-19) research as they are used to gain insight into the immune response to severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infections. Among the technologies available for generating nAbs, DNA-based immunization methods are an alternative to conventional protocols. In this pilot study, we investigated whether DNA-based immunization by needle injection in rabbits was a viable approach to produce a functional antibody response. We demonstrated that three doses of DNA plasmid carrying the gene encoding the full-length spike protein (S) or the receptor binding domain (RBD) of SARS-CoV-2 induced a time-dependent increase in IgG antibody avidity maturation. Moreover, the IgG antibodies displayed high cross neutralization by live SARS-CoV-2 and pseudoviruses neutralization assays. Thus, we established a simple, low cost and feasible DNA-based immunization protocol in rabbits that elicited high IgG avidity maturation and nAbs production against SARS-CoV-2, highlighting the importance of DNA-based platforms for developing new immunization strategies against SARS-CoV-2 and future emerging epidemics.

Implantação de um método para determinação da atividade da transcetolase eritrocitária para avaliação indireta da tiamina / Implementation of a method for determining the erythrocyte transketolase activity for indirect evaluation of thiamin

O teste de ativação da transcetolase eritrocitária (TK-E) pelo pirofosfato de tiamina (TPP) exógeno é um método indireto para mensurar a tiamina (vitamina B1). A diminuição da atividade da transcetolase eritrocitária e o aumento da estimulação in vitro com o TPP maior do que 17 % indicam deficiência de tiamina. Este é um método plausível, pois são nos eritrócitos que estão concentradas a maior parte desta vitamina. Em virtude de surtos de beribéri que tem ocorrido no Brasil desde 2006, o Instituto Adolfo Lutz (IAL), como Laboratório Central de Saúde Pública, propôs a implantação desse método para auxiliar na investigação de novos surtos ou de casos isolados. Foram avaliados o teste de precisão, a linearidade, a estabilidade do hemolisado e da amostra, e estimados os limites de detecção e de quantificação. A atividade da TK-E sem ativação pelo TPP foi de 0,732 UI/gHb e com ativação foi de 0,827 UI/gHb. Todos os resultados dos parâmetros avaliados neste estudo apresentaram-se dentro dos critérios de aceitabilidade garantindo-se a confiabilidade do método. Fica, assim, disponível mais um ensaio bioquímico para a Rede Pública de Saúde, mas ainda necessário definir os valores de referência para estabelecer os limites clínicos da deficiência de tiamina.

Erythrocyte transketolase activation test (TK-E) by exogenous thiamine pyrophosphate (TPP) is an indirect method to measure thiamine (vitamin B1). The decrease in the erythrocyte transketolase activity and the increase of in vitro stimulation with TPP greater than 17 % indicate thiamine deficiency. It is a reasonable method as the major portion of this vitamin are concentrated in erithrocytes. Due to the beriberi outbreaks that have occurred in Brazil since 2006, the Adolfo Lutz Institute (IAL), as a Central Public Health Laboratory, proposed the implementation of this method to give support to the investigation on the new outbreaks or isolated cases. The evaluated parameters were precision, linearity, hemolysate and sample stability, and the limits of detection and quantification were estimated. The TK-E activity without activation by TPP was 0.732 UI/gHb, and with activation was 0.827 UI/gHb. All of the results obtained from the evaluated parameters showed to be within the eligibility criteria, ensuring the reliability of the proposed methods. Thus, this method showed to be adequate as biochemical assay for the Public Health Network. However, there is a need to define the reference values to establish the clinical limits of thiamine deficiency.

Implantação de um método para determinação da atividade da transcetolase eritrocitária para avaliação indireta da tiamina / Implementation of a method for determining the erythrocyte transketolase activity for indirect evaluation of thiamin

O teste de ativação da transcetolase eritrocitária (TK-E) pelo pirofosfato de tiamina (TPP) exógeno é um método indireto para mensurar a tiamina (vitamina B1). A diminuição da atividade da transcetolase eritrocitária e o aumento da estimulação in vitro com o TPP maior do que 17% indicam deficiência de tiamina. Este é um método plausível, pois são nos eritrócitos que estão concentradas a maior parte desta vitamina. Em virtude de surtos de beribéri que tem ocorrido no Brasil desde 2006, o Instituto Adolfo Lutz (IAL), como Laboratório Central de Saúde Pública, propôs a implantação desse método para auxiliar na investigação de novos surtos ou de casos isolados. Foram avaliados o teste de precisão, a linearidade, a estabilidade do hemolisado e da amostra, e estimados os limites de detecção e de quantificação. A atividade da TK-E sem ativação pelo TPP foi de 0,732 UI/gHb e com ativação foi de 0,827 UI/gHb. Todos os resultados dos parâmetros avaliados neste estudo apresentaram-se dentro dos critérios de aceitabilidade garantindo-se a confiabilidade do método. Fica, assim, disponível mais um ensaio bioquímico para a Rede Pública de Saúde, mas ainda necessário definir os valores de referência para estabelecer os limites clínicos da deficiência de tiamina.

Erythrocyte transketolase activation test (TK-E) by exogenous thiamine pyrophosphate (TPP)is an indirect method to measure thiamine (vitamin B1). The decrease in the erythrocyte transketolase activity and the increase of in vitro stimulation with TPP greater than 17 % indicate thiamine deficiency. It is a reasonable method as the major portion of this vitamin are concentrated in erithrocytes. Due to the beriberi outbreaks that have occurred in Brazil since 2006, the Adolfo Lutz Institute (IAL), as a Central Public Health Laboratory, proposed the implementation of this method to give support to the investigation on the new outbreaks or isolated cases. The evaluated parameters were precision, linearity, hemolysate and sample stability, and the limits of detection and quantification were estimated. The TK-E activity without activation by TPP was 0.732 UI/gHb, and with activation was 0.827 UI/gHb. All of the results obtained from the evaluated parameters showed to be within the eligibility criteria, ensuring the reliability of the proposed methods.Thus, this method showed to be adequate as biochemical assay for the Public Health Network. However, there is a need to define the reference values to establish the clinical limits of thiamine deficiency.

Association study between genetic monoaminergic polymorphisms and OCD response to clomipramine treatment.

In the present paper, we investigated the 5HTTLPR and STin2 polymorphisms in the promoter region of the serotonin transporter gene (SLC6A4), the G861C polymorphism (rs6296) of the serotonin receptor 1D beta (HTR1B), the T102C (rs6113) and C516T (rs6305) polymorphisms of the serotonin receptor gene subtype 2A (HTR2A), the DAT UTR, DAT intron 8 and DAT intron 14 of the dopamine transporter gene (SLC6A3), the Val-158-Met (rs4680) polymorphism of the COMT and the silent mutation G1287A (rs5569) in the norepinephrine transporter gene (SLC6A2). We genotyped 41 obsessive-compulsive disorder (OCD) outpatients, classified as good-responders (n=27) and poor-responders (n=14) to treatment with clomipramine according to the Yale Brown Obsessive-Compulsive Scale (YBOCS). Patients who achieved a reduction in symptoms of 40% or more in YBOCS after 14 weeks of treatment were considered good-responders. Genotypes and alleles distribution of the investigated polymorphisms were compared between both groups. We did not find association between the studied polymorphisms and clomipramine response in our sample.

Candidate-gene approach in posttraumatic stress disorder after urban violence: association analysis of the genes encoding serotonin transporter, dopamine transporter, and BDNF.

Posttraumatic stress disorder (PTSD) is a prevalent, disabling anxiety disorder marked by behavioral and physiologic alterations which commonly follows a chronic course. Exposure to a traumatic event constitutes a necessary, but not sufficient, factor. There is evidence from twin studies supporting a significant genetic predisposition to PTSD. However, the precise genetic loci still remain unclear. The objective of the present study was to identify, in a case-control study, whether the brain-derived neurotrophic factor (BDNF) val66met polymorphism (rs6265), the dopamine transporter (DAT1) three prime untranslated region (3'UTR) variable number of tandem repeats (VNTR), and the serotonin transporter (5-HTTPRL) short/long variants are associated with the development of PTSD in a group of victims of urban violence. All polymorphisms were genotyped in 65 PTSD patients as well as in 34 victims of violence without PTSD and in a community control group (n = 335). We did not find a statistical significant difference between the BDNF val66met and 5-HTTPRL polymorphism and the traumatic phenotype. However, a statistical association was found between DAT1 3'UTR VNTR nine repeats and PTSD (OR = 1.82; 95% CI, 1.20-2.76). This preliminary result confirms previous reports supporting a susceptibility role for allele 9 and PTSD.

Association analysis between a VNTR intron 8 polymorphism of the dopamine transporter gene (SLC6A3) and obsessive- compulsive disorder in a Brazilian sample.

Family, twin and segregation analysis have provided evidences that genetic factors are implicated in the susceptibility for obsessive-compulsive disorder (OCD). Several lines of research suggest that the dopaminergic system may be involved in the pathophysiology of OCD. Thus, the aim of the present study was to investigate a possible association between a polymorphism located in intron 8 of the dopamine transporter gene (SLC6A3) and OCD in a Brazilian sample composed by 208 patients and 865 healthy controls. No statistical differences were observed in allelic and genotype distributions between cases and controls. No association was also observed when the sample was divided according to specific phenotypic features such as gender, presence of tic disorders, co-morbidity, and age at onset of obsessive-compulsive symptoms (OCS). Our results suggest that the intron 8 VNTR of the SLC6A3 investigated in this study is not related to the susceptibility for OCD in our Brazilian sample.

Association analysis between a VNTR intron 8 polymorphism of the dopamine transporter gene (SLC6A3) and obsessive- compulsive disorder in a Brazilian sample

Family, twin and segregation analysis have provided evidences that genetic factors are implicated in the susceptibility for obsessive-compulsive disorder (OCD). Several lines of research suggest that the dopaminergic system may be involved in the pathophysiology of OCD. Thus, the aim of the present study was to investigate a possible association between a polymorphism located in intron 8 of the dopamine transporter gene (SLC6A3) and OCD in a Brazilian sample composed by 208 patients and 865 healthy controls. No statistically differences were observed in allelic and genotype distributions between cases and controls. No association was also observed when the sample was divided according to specific phenotypic features such as gender, presence of tic disorders co-morbidity and age at onset of obsessive-compulsive symptoms (OCS). Our results suggest that the intron 8 VNTR of the SLC6A3 investigated in this study is not related to the susceptibility for OCD in our Brazilian sample.

Estudos de família, gêmeos e de segregação têm demonstrado que fatores genéticos estão envolvidos na susceptibilidade para o desenvolvimento do transtorno obsessivo-compulsivo (TOC). Várias linhas de pesquisa sugerem que o sistema dopaminérgico possa estar envolvido na fisiopatologia do TOC. Assim, o objetivo do presente estudo foi investigar uma possível associação entre o polimorfismo localizado no intron 8 do gene do transportador da dopamina (SLC6A3) e o TOC em uma amostra brasileira composta por 208 pacientes e 865 controles sadios. Nenhuma diferença estatisticamente significante foi observada nas distribuições alélicas e genotípicas entre os grupos de pacientes e controles. Nenhuma associação também foi observada quando as amostras foram divididas de acordo com características fenotípicas específicas, tais como gênero, presença de co-morbidade com tiques e idade de início dos sintomas obsessivo-compulsivo (SOC). Nossos resultados sugerem que o VNTR do intron 8 investigado neste estudo não está relacionado com o TOC na nossa amostra brasileira.

Riboflavin deficiency in pregnant womem in a public institution of São Paulo city, SP, Brasil

vitamin doses were given and in women who did not receive any vitamin supplementation during gestation. Riboflavin nutritional determination was assessed by means of glutathione reductase activation coefficient (GR-AC), and the value higher than 1.5 was considered as indicative of riboflavin deficiency. The pregnant women were divided into four groups: 123 pregnant women to whom any vitamin supplementation were not given, 25 who daily received 2.5 to 3.5mg of riboflavin, 53 who were daily supplemented with 1.0 to 1.7mg of riboflavin, and 22 to whom were daily given <0.85mg of riboflavin. The women group daily supplemented with 2.5 to 3.5mg riboflavin showed the lowest riboflavin deficiency, and the other groups (64.5%) presented vitamin deficiency with GR-AC higher than 1.5. These data suggest that the vitamin concentrations found in the commercially available vitamin supplemented – preparations, as well as those found in ingested food were insufficient to provide a good nutritional status for pregnant women. These findings strongly suggest that riboflavin deficiency has in fact been a relevant public health problem in São Paulo city.

Neste estudo prospectivo foi avaliado o estado nutricional em riboflavina nas parturientes que faziam uso da suplementação vitamínica de variadas concentrações de riboflavina, bem como naquelas que não fizeram uso de qualquer suplementação durante o período gestacional. O recurso utilizado para a avaliação nutricional foi a determinação do coeficiente de ativação da glutationa redutase (CA-GRE). Os valores de CA-GRE acima de 1,5 foram considerados como indicativos de deficiência de riboflavina. Foram analisados quatro grupos de gestantes: 123 parturientes sem suplementação vitamínica; 25 que utilizaram formulação contendo de 2,5 a 3,5mg de riboflavina; 63 parturientes que utilizaram fármacos contendo 1,0 a 1,7mg de riboflavina e o quarto grupo constituído de 22 mulheres que receberam formulação contendo < 0,85mg de riboflavina. O grupo de parturientes que apresentou menor índice de deficiência de riboflavina foi aquele que fez o uso da suplementação vitamínica com as taxas entre 2,5 a 3,5mg de vitamina B2. Os demais grupos, com a inclusão daquele que não complementou a dieta com suplementação vitamínica, apresentaram índices de deficiências bem maiores. Esses achados indicam que as quantidades de riboflavina apresentadas nesses fármacos, bem como na dieta alimentar, foram insuficientes para atingir níveis bioquímicos semelhantes ao do grupo controle. No geral, foi encontrada uma incidência de deficiência de riboflavina de 64,4%, o que permite sugerir que a arriboflavinose é um importante problema de Saúde Pública na cidade de São Paulo.

Avaliação da deficiência de riboflavina em gestantes de uma instituição pública da cidade de São Paulo, SP, Brasil / Avaliação da deficiência de riboflavina em gestantes de uma instituição pública da cidade de São Paulo, SP, Brasil

In a prospective study, the riboflavin nutritional status was evaluated in pregnant women to whom varied vitamin doses were given and in women who did not receive any vitamin supplementation during gestation. Riboflavin nutritional determination was assessed by means of glutathione reductase activation coefficient (GR-AC), and the value higher than 1.5 was considered as indicative of riboflavin deficiency. The pregnant women were divided into four groups: 123 pregnant women to whom any vitamin supplementation were not given, 25 who daily received 2.5 to 3.5mg of riboflavin, 53 who were daily supplemented with 1.0 to 1.7mg of riboflavin, and 22 to whom were daily given 0.85mg of riboflavin. The women group daily supplemented with 2.5 to 3.5mg riboflavin showed the lowest riboflavin deficiency, and the other groups (64.5%) presented vitamin deficiency with GR-AC higher than 1.5. These data suggest that the vitamin concentrations found in the commercially available vitamin supplemented preparations, as well as those found in ingested food were insufficient to provide a good nutritional status for pregnant women. These findings strongly suggest that riboflavin deficiency has in fact been a relevant public health problem in São Paulo city.

Neste estudo prospectivo foi avaliado o estado nutricional em riboflavina nas parturientes que faziam uso da suplementação vitamínica de variadas concentrações de riboflavina, bem como naquelas que não fizeram uso de qualquer suplementação durante o período gestacional. O recurso utilizado para a avaliação nutricional foi a determinação do coeficiente de ativação da glutationa redutase (CA-GRE). Os valores de CA-GRE acima de 1,5 foram considerados como indicativos de deficiência de riboflavina. Foram analisados quatro grupos de gestantes: 123 parturientes sem suplementação vitamínica; 25 que utilizaram formulação contendo de 2,5 a 3,5mg de riboflavina; 63 parturientes que utilizaram fármacos contendo 1,0 a 1,7mg de riboflavina e o quarto grupo constituído de 22 mulheres que receberam formulação contendo 0,85mg de riboflavina. O grupo de parturientes que apresentou menor índice de deficiência de riboflavina foi aquele que fez o uso da suplementação vitamínica com as taxas entre 2,5 a 3,5mg de vitamina B2. Os demais grupos, com a inclusão daquele que não complementou a dieta com suplementação vitamínica, apresentaram índices de deficiências bem maiores. Esses achados indicam que as quantidades de riboflavina apresentadas nesses fármacos, bem como na dieta alimentar, foram insuficientes para atingir níveis bioquímicos semelhantes ao do grupo controle. No geral, foi encontrada

Investigation of possible association between Ser9Gly polymorphism of the D3 dopaminergic receptor gene and response to typical antipsychotics in patients with schizophrenia.

Typical antipsychotics have a high affinity for dopamine receptors. It is therefore of interest to investigate such loci in pharmacogenetic studies on psychosis. We investigated the hypothesis that Ser9Gly polymorphism of the DRD3 gene may play a role in the differences in individual response to typical antipsychotics between schizophrenic patients. The sample was composed of 53 good responders and 59 poor ones. No significant differences between the good and poor responders were found in the allelic distribution (good responders: Ser9 61.32%, Gly9 38.67%; poor responders: Ser9 64.40%, Gly9 35.59%; odds ratio, OR = 0.88, 0.49 < OR < 1.56; chi2 = 0.23, 1 degree of freedom, df, p = 0.63) and genotype distribution (good responders: Ser9/Ser9 37.73%, Ser9/Gly9 47.16%, Gly9/Gly9 15.09%; poor responders: Ser9/Ser9 42.37%, Ser9/Gly9 44.06%, Gly9/Gly9 13.55%; chi2 = 0.25, 2 df, p = 0.88). Nor was there any association with homozygosity (good responders: homozygous: 52.82%, heterozygous: 47.16%; poor responders: homozygous: 55.92%, heterozygous: 44.06%; odds ratio, OR = 0.88, 0.39 < OR < 1.99; chi2 = 0.11, 1 df, p = 0.74). The results did not support the hypothesis that Ser9Gly polymorphism of the DRD3 gene influences the response to typical antipsychotics in our sample of schizophrenics.

Association study of the INPP1, 5HTT, BDNF, AP-2beta and GSK-3beta GENE variants and restrospectively scored response to lithium prophylaxis in bipolar disorder.

In the present study we investigated the influence of a series variants in genes (the serotonin transporter, glycogen synthase kinase-3beta, inositol polyphosphatase 1-phosphate, brain-derived neurotrophic factor and activator protein 2beta) related to the action of lithium carbonate, a drug used for prophylaxis in mood disorders. We used a sample of unrelated patients with bipolar disorder type I on lithium therapy for at least 2 years who met the proposed response criteria for prophylactic response. Of the 134 patients, 61 patients were considered full responders, 49 non-responders and 24 partial responders. No significant differences were observed for the genotype or allele frequencies for good, partial and poor responders for the five gene variants: for BDNF G196A (genotype: chi2 = 3.67, 4 d.f., p = 0.45; allele: chi2 = 2.31, 2 d.f., p = 0.31); for INPP1 C973A (genotype: chi2 = 1.35, 4 d.f., p = 0.85; allele: chi2 = 0.04, 2 d.f., p = 0.98); for AP-2beta [CAAA](4/5) (genotype: chi2 = 3.18; 4 d.f., p = 0.52; allele: chi2 = 0.92, 2 d.f., p = 0.063); for 5HTTLPR (genotype: chi2 = 0.67, 4 d.f., p = 0.96; allele: chi2 = 0.27, 2 d.f., p = 0.87); for GSK-3beta A-1727T (genotype: chi2 = 3.55, 4 d.f., p = 0.47; allele: chi2 = 0.48, 2 d.f., p = 0.78). These investigated variants are not predictive factors for lithium prophylactic response in our sample of bipolar disorder type I patients. However, it is still possible that a subgroup of a diverse ethnic ancestry may be predisposing to some of those variants for lithium response.

Investigation of possible association between Ser9Gly polymorphism of the D3 dopaminergic receptor gene and response to typical antipsychotics in patients with schizophrenia / Investigação da possível associação entre o polimorfismo Ser9Gly do gene do receptor de dopamina D3 e resposta a antipsicóticos típicos em pacientes com esquizofrenia

Typical antipsychotics have a high affinity for dopamine receptors. It is therefore of interest to investigate such loci in pharmacogenetic studies on psychosis. We investigated the hypothesis that Ser9Gly polymorphism of the DRD3 gene may play a role in the differences in individual response to typical antipsychotics between schizophrenic patients. The sample was composed of 53 good responders and 59 poor ones. No significant differences between the good and poor responders were found in the allelic distribution (good responders: Ser9 61.32 percent, Gly9 38.67 percent; poor responders: Ser9 64.40 percent, Gly9 35.59 percent; odds ratio, OR = 0.88, 0.49 < OR < 1.56; chi2 = 0.23, 1 degree of freedom, df, p = 0.63) and genotype distribution (good responders: Ser9/Ser9 37.73 percent, Ser9/Gly9 47.16 percent, Gly9/Gly9 15.09 percent; poor responders: Ser9/Ser9 42.37 percent, Ser9/Gly9 44.06 percent, Gly9/Gly9 13.55 percent; chi2 = 0.25, 2 df, p = 0.88). Nor was there any association with homozygosity (good responders: homozygous: 52.82 percent, heterozygous: 47.16 percent; poor responders: homozygous: 55.92 percent, heterozygous: 44.06 percent; odds ratio, OR = 0.88, 0.39 < OR < 1.99; chi2 = 0.11, 1 df, p = 0.74). The results did not support the hypothesis that Ser9Gly polymorphism of the DRD3 gene influences the response to typical antipsychotics in our sample of schizophrenics.

Antipsicóticos típicos apresentam alta afinidade pelos receptores dopaminérgicos, que são, portanto, regiões de interesse para os estudos de farmacogenética das psicoses. No presente estudo, investigamos a hipótese de que o polimorfismo Ser9Gly do gene do DRD3 possa exercer um papel na diferença de resposta inter-individual ao uso de antipsicóticos típicos em pacientes com esquizofrenia. Nossa amostra foi composta por 53 pacientes bons respondedores e 59 maus respondedores. Não houve diferenças nas distribuições alélicas (bons respondedores: Ser9 61,32 por cento, Gly9 38,67 por cento; maus respondedores: Ser9 64,40 por cento, Gly9 35,59 por cento; odds ratio, OR = 0,88, 0,49 < OR < 1,56; chi2 = 0,23, 1 degree of freedom, d.f., p = 0,63) e genotípica (bons respondedores: Ser9/Ser9 37,73 por cento, Ser9/Gly9 47,16 por cento, Gly9/Gly9 15,09 por cento; maus repondedores: Ser9/Ser9 42,37 por cento, Ser9/Gly9 44,06 por cento, Gly9/Gly9 13,55 por cento; chi2 = 0,25, 2 d.f., p = 0,88) entre os grupos. Não houve também associação com homozigosidade (bons respondedores: homozigotos: 52,82 por cento, heterozigotos: 47,16 por cento; maus repondedores: homozigotos: 55,92 por cento, heterozigotos: 44,06 por cento; odds ratio, OR = 0,88, 0,39 < OR < 1,99); chi2 = 0,11, 1 d.f., p = 0,74). Os resultados não dão suporte à hipótese de que o polimorfismo Ser9Gly do gene do DRD3 possa influenciar a resposta terapêutica aos antipsicóticos típicos na nossa amostra de pacientes com esquizofrenia.