RESUMO
Participation in an EQA program is critical to the quality assurance process. Reliable and precise CD4 T-cells enumeration are essential to improve the clinical management of patients by evaluating the disease progression and by monitoring the effectiveness of ART in HIV-patients. The CIRCB, CD4 reference laboratory, in collaboration with the Canadian QASI-program, recruited sites, distributed and analyzed CD4-panels in 61 sites across Cameroon. A trend and performance analysis in the pre-analytical, analytical and post-analytical phases was performed. Continuous training and corrective actions carried out from 2014 to 2018 increased the number of participating sites from 15 to 61 sites, the number of unacceptable results decreased from 50 to 10%. Specific challenges included errors in pre analytic (17.5%), analytic (77.0%) and post-analytic (5.5%) phases. This EQA requires the application of good laboratory practices, fluidic communication between all the stakeholders, continuous training, application of specific on-site corrective measures, and timely equipment maintenance in order to avoid repetitive errors and to increase laboratory performance. It could be extended to other HIV-1 testing like viral load and EID point-of-care. Partnership with QASI serve as a model for implementation of a successful EQA model for resource limited countries wanting to implement EQA for HIV testing and monitoring in alignment with 90-90-90 targets.
RESUMO
BACKGROUND: CD4 T cell enumeration is the most widely used prognostic marker for management of HIV disease. Internal quality control and external quality assessment (EQA) programs are critical to ensure reliability of clinical measurements. The utility of stabilized whole blood products (SWBP) as a test reagent for EQA programs such as Quality Assessment and Standardization for Immunological measures relevant to HIV/AIDS (QASI) program have been demonstrated previously. Since then, several new commercial SWBPs and alternative CD4 enumeration technologies have become available. Seven SWBPs were evaluated on seven different enumeration platforms to determine which product(s) are most suitable for EQA programs that support multiple analytical technologies. METHOD: Assessment of SWBPs was based on two criteria: (1) accuracy of CD4 T cell measurements and; (2) stability under sub optimal storage conditions. RESULTS: Three SWBPs (Multi-Check, StatusFlow and CD4 Count) showed accurate CD4 T-cell absolute count and percentage values across six of the enumeration platforms. All products retain stability up to 18 days at 21-23°C with the exception of Multi-Check-high on FacsCount and Multi-Check-Low and StatusFlow-Low on Pima. One of the products (CD4 Count) retained stability for three days on all platforms tested when stored at 37°C. CONCLUSION: This study demonstrated that the characteristics of commercially available SWBPs vary across multiple CD4 platforms. The compatibility of testing panels for EQA programs with multiple analytical platforms needs to be carefully considered, especially in large multiplatform CD4 EQA programs. The selection of a suitable cross-platform SWBP is an increasing challenge as more reagents and platforms are introduced for CD4 T-cell enumeration.
Assuntos
Produtos Biológicos , Sangue/imunologia , Linfócitos T CD4-Positivos/citologia , Contagem de Células/métodos , Soropositividade para HIV/sangue , Soropositividade para HIV/diagnóstico , Produtos Biológicos/química , Contagem de Células/normas , Humanos , Imunofenotipagem/métodos , Controle de QualidadeRESUMO
A subset of women in the Pumwani Sex Worker Cohort, established in 1985 in Nairobi, Kenya, remains uninfected despite repeated high-risk exposure (HIV-exposed, seronegative [HESN]) through active sex work. This HESN phenotype is associated with several alleles of human leukocyte antigens (HLAs) and specific CD8(+) and CD4(+) T cell responses to HIV-1. The associations of HLA alleles with differential HIV-1 infection are most likely due to their different abilities to present antigen and the different immune responses they induce. The characteristics of epitopes of HLA alleles associated with different outcomes of HIV-1 infection might therefore point to a vital clue for developing an effective vaccine. In this study, we systematically analyzed HIV-1 clade A and D Gag CD8(+) T cell epitopes of two HLA class I alleles associated with different outcomes of HIV-1 infection. Binding affinity and off-rates of the identified epitopes were determined. Gamma interferon (IFN-γ) enzyme-linked immunospot (ELISpot) assays with patient peripheral blood mononuclear cells (PBMCs) validated the epitopes. Epitope-specific CD8(+) T cells were further phenotyped for memory markers with tetramer staining. Our study showed that the protective allele A*01:01 recognizes only three Gag epitopes. By contrast, B*07:02, the allele associated with susceptibility, binds 30 epitope variants. These two alleles differ most importantly in the spectrum of Gag epitopes they can present and not in affinity, off-rates, the location of the epitopes, or epitope-specific Tem/Tcm frequencies. The binding of more epitopes and strong IFN-gamma ELISpot responses are associated with susceptibility to HIV-1 infection, while more focused antigen recognition of multiple subtypes is protective. Rational vaccine design should take these observations into account.
Assuntos
Epitopos de Linfócito T/imunologia , Infecções por HIV/genética , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Antígenos HLA/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia , Adulto , Alelos , Sequência de Aminoácidos , Estudos de Coortes , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Feminino , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/química , HIV-1/genética , HIV-1/fisiologia , Antígenos HLA/imunologia , Humanos , Quênia , Dados de Sequência Molecular , Alinhamento de Sequência , Profissionais do Sexo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genéticaRESUMO
BACKGROUND: Malaria and schistosomiasis coinfection frequently occurs in tropical countries. This study evaluates the influence of Schistosoma haematobium infection on specific antibody responses and cytokine production to recombinant merozoite surface protein-1-19 (MSP1-(19)) and schizont extract of Plasmodium falciparum in malaria-infected children. METHODOLOGY: Specific IgG1 to MSP1-(19), as well as IgG1 and IgG3 to schizont extract were significantly increased in coinfected children compared to P. falciparum mono-infected children. Stimulation with MSP1-(19) lead to a specific production of both interleukin-10 (IL-10) and interferon-γ (IFN-γ), whereas the stimulation with schizont extract produced an IL-10 response only in the coinfected group. CONCLUSIONS: Our study suggests that schistosomiasis coinfection favours anti-malarial protective antibody responses, which could be associated with the regulation of IL-10 and IFN-γ production and seems to be antigen-dependent. This study demonstrates the importance of infectious status of the population in the evaluation of acquired immunity against malaria and highlights the consequences of a multiple infection environment during clinical trials of anti-malaria vaccine candidates.
Assuntos
Anticorpos Anti-Helmínticos/imunologia , Antígenos de Protozoários/imunologia , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Esquistossomose Urinária/imunologia , Adolescente , Animais , Anticorpos Antiprotozoários/imunologia , Criança , Citocinas/imunologia , Humanos , Malária Falciparum/complicações , Malária Falciparum/parasitologia , Masculino , Proteína 1 de Superfície de Merozoito/imunologia , Plasmodium falciparum/fisiologia , Schistosoma haematobium/imunologia , Schistosoma haematobium/fisiologia , Esquistossomose Urinária/complicações , Esquistossomose Urinária/parasitologiaRESUMO
Members of the protein kinase C (PKC) family are activated by interferon-gamma (IFN-gamma) and modulate IFN-gamma-induced cellular responses by regulating the activity of transcription factors. We previously reported that PKC-alpha enhances the ability of IFN regulatory factor-1 to transactivate the class II transactivator (CIITA) promoter IV in IFN-gamma-stimulated macrophages. In addition, we showed that IFN-gamma induces the nuclear translocation of PKC-alpha but the mechanisms for this remain to be elucidated. In this study, we sought to identify signalling pathways involved in IFN-gamma-induced activation of PKC-alpha and to characterize their potential roles in modulating IFN-gamma-induced responses in macrophages. IFN-gamma-mediated nuclear translocation of PKC-alpha was a Janus activated kinase 2 (JAK2)-independent process, which required phosphatidylinositol 3-kinase (PI3K) and p38 mitogen-activated protein kinase (MAPK). However, PKC-alpha phosphorylation was independent of PI3K and p38 MAPK, indicating that IFN-gamma-induced phosphorylation and nuclear translocation of PKC-alpha are mediated by distinct mechanisms. In addition, inhibition of PI3K, but not of p38 MAPK, strongly impaired IFN-gamma-induced CIITA and MHC II gene expression. Finally, PKC-alpha associated with signal transducer and activator of transcription 1 (STAT1) and was required for the phosphorylation of STAT1 on serine 727 in IFN-gamma-stimulated macrophages. Taken together, our data indicate that PI3K and p38 MAPK modulate IFN-gamma-stimulated PKC-alpha nuclear translocation independently of JAK2 activity and that both PI3K and PKC-alpha are required for type IV CIITA and MHC II gene expression in IFN-gamma-stimulated macrophages.
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Núcleo Celular/enzimologia , Macrófagos/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Quinase C-alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Linhagem Celular , Cromonas/farmacologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Feminino , Imidazóis/farmacologia , Interferon gama/farmacologia , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Morfolinas/farmacologia , Proteínas Nucleares/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteína Quinase C-alfa/antagonistas & inibidores , Piridinas/farmacologia , Fator de Transcrição STAT1/efeitos dos fármacos , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Transativadores/efeitos dos fármacos , Transativadores/metabolismo , Tirfostinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Phagocytosis of Leishmania donovani promastigotes is characterized by an inhibition of phagolysosome biogenesis mediated by the surface glycolipid lipophosphoglycan (LPG). However, the consequences of this inhibition on macrophage function remain to be determined. In this study, we investigated the impact of LPG-mediated phagosome remodelling on the assembly and function of the NADPH oxidase complex. Phagocytosis of both wild-type and LPG-defective L. donovani promastigotes triggered the release of similar levels of superoxide. However, wild-type promastigotes, but not LPG-defective mutants, inhibited generation of superoxide at the phagosome. Confocal microscopy imaging revealed that the membrane component gp91(phox) and the Rho-family GTPase Rac1 were present on phagosomes containing either wild-type or LPG-defective promastigotes. In contrast, the NADPH oxidase cytosolic components p47(phox) and p67(phox) were excluded from phagosomes in a LPG-dependent fashion. This inhibition is not the consequence of a general defect in the initiation of the NADPH oxidase activation process because both wild-type and LPG-defective promastigotes induced p47(phox) phosphorylation and the formation of complexes containing p47(phox) and p67(phox). Thus, by remodelling their intracellular habitat, L. donovani promastigotes prevent the assembly of a functional phagosomal NADPH oxidase complex, thereby evading an important host innate defence mechanism.
Assuntos
Glicoesfingolipídeos/metabolismo , Leishmania donovani/patogenicidade , Macrófagos Peritoneais/parasitologia , NADPH Oxidases/metabolismo , Fagossomos/metabolismo , Animais , Feminino , Membranas Intracelulares/metabolismo , Leishmania donovani/química , Leishmania donovani/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Fagocitose , Fagossomos/enzimologia , Fosfoproteínas/metabolismo , Superóxidos/metabolismoRESUMO
This study has evaluated the individual control of isotype production and the influence of external signals that can be experimentally provided in vitro, in antibody responses to two different recombinant Schistosoma antigens (Sh28GST and TPx-1). Peripheral blood mononuclear cells or enriched B cell fractions obtained from S. haematobium infected Senegalese adults were induced to terminal differentiation in vitro. The production of antibody to either antigen was donor-dependent and for each donor it was antigen-dependent. Differentiation to IgG1 and IgG3 production, and possibly IgA, specific to these conserved parasite antigens could be regulated differentially in vitro. Exogenous IL-2 and IL-10 or IL-10 and TGF-beta led to the production of specific IgG3 or IgG1 and/or IgA, respectively. This is the first report on such experimentally induced differential regulation of antigen-specific IgG1 and IgG3. This may have implications in designing protocols for protein based-vaccinations aiming at eliciting antibody responses of certain protective-type isotypes.
Assuntos
Anticorpos Anti-Helmínticos/biossíntese , Antígenos de Helmintos/imunologia , Imunoglobulina A/biossíntese , Imunoglobulina G/biossíntese , Schistosoma haematobium/imunologia , Esquistossomose Urinária/imunologia , Adolescente , Animais , Anticorpos Anti-Helmínticos/sangue , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/metabolismo , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Feminino , Humanos , Imunoglobulina A/sangue , Imunoglobulina A/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Interleucina-10/farmacologia , Interleucina-2/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade , Contagem de Ovos de Parasitas , Schistosoma haematobium/crescimento & desenvolvimento , Esquistossomose Urinária/epidemiologia , Fator de Crescimento Transformador beta/farmacologiaRESUMO
This study examined the evolution of immunoglobulin (Ig) G1 and IgG3 antibodies against the asexual stage Plasmodium falciparum protein, MSP1(19), before and after a heavy malaria transmission period in clinically immune Senegalese subjects living under different epidemiological conditions. Plasma was tested for antibodies to a yeast-produced, recombinant PfMSP1(19) antigen (the Q-KNG allelic variant) that has previously been demonstrated to react with IgG1, IgG3, or both in the majority of these people. Anti-P. falciparum antibodies of the IgG1 and IgG3 subclasses, previously reported to be associated with protection, were shown to evolve independently one from another after the transmission period in both settings. These results suggest differential regulation of MSP1(19)-specific IgG1 and IgG3. The precise role of these antibody isotypes in maintaining malaria immunity remains to be determined.