25 research outputs found
Immunoglobulin signal transduction guides the specificity of B cell-T cell interactions and is blocked in tolerant self-reactive B cells.
The specificity of antibody (Ab) responses depends on focusing helper T (Th) lymphocyte signals to suitable B lymphocytes capable of binding foreign antigens (Ags), and away from nonspecific or self-reactive B cells. To investigate the molecular mechanisms that prevent the activation of self-reactive B lymphocytes, the activation requirements of B cells specific for the Ag hen egg lysozyme (HEL) obtained from immunoglobulin (Ig)-transgenic mice were compared with those of functionally tolerant B cells isolated from Ig-transgenic mice which also express soluble HEL. To eliminate the need for surface (s)Ig-mediated Ag uptake and presentation and allow the effects of sIg signaling to be studied in isolation, we assessed the ability of allogeneic T cells from bm12 strain mice to provide in vivo help to C57BL/6 strain-transgenic B cells. Interestingly, non-tolerant Ig-transgenic B cells required both allogeneic Th cells and binding of soluble HEL for efficient activation and Ab production. By contrast, tolerant self-reactive B cells from Ig/HEL double transgenic mice responded poorly to the same combination of allogeneic T cells and soluble HEL. The tolerant B cells were nevertheless normally responsive to stimulation with interleukin 4 and anti-CD40 Abs in vitro, suggesting that they retained the capacity to respond to mediators of T cell help. However, the tolerant B cells exhibited a proximal block in the sIg signaling pathway which prevented activation of receptor-associated tyrosine kinases in response to the binding of soluble HEL. The functional significance of this sIg signaling defect was confirmed by using a more potent membrane-bound form of HEL capable of triggering sIg signaling in tolerant B cells, which markedly restored their ability to collaborate with allogeneic Th cells and produce Ab. These findings indicate that Ag-specific B cells require two signals for mounting a T cell-dependent Ab response and identify regulation of sIg signaling as a mechanism for controlling self-reactive B cells
Comportamento de células do sistema imune frente ao desafio com Salmonella Enteritidis em aves tratadas e não tratadas com ácidos orgânicos
A Salmonelose é uma importante zoonose, considerada a principal causa de infecções bacterianas, sendo associada ao consumo de produtos avícolas. Como alternativa de controle, ácidos orgânicos têm sido amplamente usados. No entanto, pouco se conhece sobre o estado imunológico de aves de produção, e uma avaliação deste status é necessária para proteger frente a enfermidades e para garantir à aplicação segura de agentes terapêuticos ou imunização profilática. Este trabalho teve como objetivo verificar o comportamento do sistema imunológico das aves previamente infectadas com Salmonella Enteritidis (SE) tratadas com um composto de ácidos orgânicos em diferentes concentrações administrado via água e ração comparando com as aves infectadas e não tratadas. Foram inoculados 120 frangos de corte com 1mL de SE, via oral, na concentração de 1,0 x 108 UFC/mL, no 1º e 2º dia de idade, divididos em seis tratamentos com duas repetições, utilizando 200, 400, 500 e 1000ppm do ácido orgânico. Aos 35 dias de vida das aves, foram coletados, de todos os grupos, alíquotas de sangue de 3mL em tubo contendo EDTA para a avaliação das células imunes através de citometria de fluxo. Foram analisadas as porcentagens circulantes de células CD4+, CD8β+, MHC I+, MHC II+, TCRVβ1+, TCRVβ2+ e CD28+. Para análise microbiológica foram coletadas tonsilas cecais destas aves. Observou-se com esse estudo que os ácidos orgânicos nas dosagens 1000ppm na água e 500ppm na ração durante, dois e sete dias respectivamente antes do abate, foram eficazes na redução da infecção por SE em frangos de corte, comprovadas pelo método microbiológico e demonstradas através do comportamento das células do sistema imune. No presente estudo as aves infectadas apresentaram uma proporção menor de células T auxiliares circulantes quando comparadas às aves infectadas, mas tratadas com o AO ou com o grupo não infectado. A mesma tendência pode ser observada para as células CD28+, TCRVβ1+ e MHC IIbright+, e, com menor resolução, para CD8β+
A large-scale RNAi screen in human cells identifies new components of the p53 pathway
RNA interference (RNAi) is a powerful new tool with which to
perform loss-of-function genetic screens in lower organisms and
can greatly facilitate the identification of components of cellular
signalling pathways. In mammalian cells, such screens have
been hampered by a lack of suitable tools that can be used on a
large scale. We and others have recently developed expression
vectors to direct the synthesis of short hairpin RNAs (shRNAs)
that act as short interfering RNA (siRNA)-like molecules to stably
suppress gene expression. Here we report the construction of a
set of retroviral vectors encoding 23,742 distinct shRNAs, which
target 7,914 different human genes for suppression. We use this
RNAi library in human cells to identify one known and five new
modulators of p53-dependent proliferation arrest. Suppression
of these genes confers resistance to both p53-dependent and
p19ARF-dependent proliferation arrest, and abolishes a DNAdamage-
induced G1 cell-cycle arrest. Furthermore, we describe
siRNA bar-code screens to rapidly identify individual siRNA
vectors associated with a specific phenotype. These new tools
will greatly facilitate large-scale loss-of-function genetic screens
in mammalian cells
Construction and Characterization of a Recombinant Vaccinia Virus Expressing Murine Intercellular Adhesion Molecule-1: Induction and Potentiation of Antitumor Responses
Gene expression profiling predicts clinical outcome of breast cancer
Breast cancer patients with the same stage of disease can have
markedly different treatment responses and overall outcome. The
strongest predictors for metastases (for example, lymph node
status and histological grade) fail to classify accurately breast
tumours according to their clinical behaviour. Chemotherapy
or hormonal therapy reduces the risk of distant metastases by
approximately one-third; however, 70 — 80% of patients receiving
this treatment would have survived without it. None of the
signatures of breast cancer gene expression reported to date
allow for patient-tailored therapy strategies. Here we used DNA
microarray analysis on primary breast tumours of 117 young
patients, and applied supervised classification to identify a gene
expression signature strongly predictive of a short interval to
distant metastases ('poor prognosis' signature) in patients without
tumour cells in local lymph nodes at diagnosis (lymph node
negative). In addition, we established a signature that identifies
tumours of BRCAI carriers. The poor prognosis signature consists
of genes regulating cell cycle, invasion, metastasis and angiogenesis. This gene expression profile will outperform all
currently used clinical parameters in predicting disease outcome.
Our findings provide a strategy to select patients who would
benefit from adjuvant therapy
Minimizing the risk of reporting false positives in large-scale RNAi screens
Large-scale RNA interference (RNAi)-based analyses, very much as other ‘omic’ approaches, have inherent rates of false positives and negatives. The variability in the standards of care applied to validate results from these studies, if left unchecked, could eventually begin to undermine the credibility of RNAi as a
powerful functional approach. This Commentary is an invitation to an open discussion started among various users of RNAi to set forth accepted standards that would insure the quality and accuracy of
information in the large datasets coming out of genome-scale screens