A series of Fas receptor agonist antibodies that demonstrate an inverse correlation between affinity and potency

Receptor agonism remains poorly understood at the molecular and mechanistic level. In this study, we identified a fully human anti-Fas antibody that could efficiently trigger apoptosis and therefore function as a potent agonist. Protein engineering and crystallography were used to mechanistically understand the agonistic activity of the antibody. The crystal structure of the complex was determined at 1.9 Å resolution and provided insights into epitope recognition and comparisons with the natural ligand FasL (Fas ligand). When we affinity-matured the agonist antibody, we observed that, surprisingly, the higher-affinity antibodies demonstrated a significant reduction, rather than an increase, in agonist activity at the Fas receptor. We propose and experimentally demonstrate a model to explain this non-intuitive impact of affinity on agonist antibody signalling and explore the implications for the discovery of therapeutic agonists in general

FasL is more frequently expressed in liver metastases of colorectal cancer than in matched primary carcinomas

Colorectal carcinoma cells have recently been shown to express Fas ligand (FasL). This ligand could allow the tumour cells to evade activated tumour-infiltrating lymphocytes (TILs) by inducing their apoptosis and would thus promote tumour survival and possibly metastasis formation. To test this hypothesis in vivo we analysed the expression of FasL mRNA and protein in paired tissue samples of normal colonic mucosa (N), primary colorectal carcinomas (T) and their metastases (M) from a total of 21 patients by four different methods. Additionally, the presence and activation status of infiltrating lymphocytes, which might contribute to the total amount of FasL in the tissue, was determined by semiquantitative reverse transcription–polymerase chain reaction (RT–PCR) in the same samples. The frequency of FasL detection was 30–40% in T and was 60–100% in M, depending on the sensitivity of the method. Simultaneously, the amount of CD25 mRNA, used as a measure of the number of activated TILs, was in 90% of patients lower in M than in T. The increased frequency of FasL detection in liver metastases was therefore not due to the presence of activated TILs. We conclude that metastasizing subpopulations of colorectal tumour cells express FasL more frequently than the primary carcinomas and may be able to eliminate activated TILs in vivo via Fas/FasL-induced apoptosis or other hitherto unknown mechanisms. © 1999 Cancer Research Campaig

Program design features that can improve participation in health education interventions

<p>Abstract</p> <p>Background</p> <p>Although there have been reported benefits of health education interventions across various health issues, the key to program effectiveness is participation and retention. Unfortunately, not everyone is willing to participate in health interventions upon invitation. In fact, health education interventions are vulnerable to low participation rates. The objective of this study was to identify design features that may increase participation in health education interventions and evaluation surveys, and to maximize recruitment and retention efforts in a general ambulatory population.</p> <p>Methods</p> <p>A cross-sectional questionnaire was administered to 175 individuals in waiting rooms of two hospitals diagnostic centres in Toronto, Canada. Subjects were asked about their willingness to participate, in principle, and the extent of their participation (frequency and duration) in health education interventions under various settings and in intervention evaluation surveys using various survey methods.</p> <p>Results</p> <p>The majority of respondents preferred to participate in one 30–60 minutes education intervention session a year, in hospital either with a group or one-on-one with an educator. Also, the majority of respondents preferred to spend 20–30 minutes each time, completing one to two evaluation surveys per year in hospital or by mail.</p> <p>Conclusion</p> <p>When designing interventions and their evaluation surveys, it is important to consider the preferences for setting, length of participation and survey method of your target population, in order to maximize recruitment and retention efforts. Study respondents preferred short and convenient health education interventions and surveys. Therefore, brevity, convenience and choice appear to be important when designing education interventions and evaluation surveys from the perspective of our target population.</p

Superantigen reactive Vβ6+ T cells induce perforin/granzyme B mediated caspase-independent apoptosis in tumour cells

The endogenous viral superantigen 7 in DBA/2 mice serves as a target antigen on syngeneic ESb-MP lymphoma cells for allogeneic graft-vs-leukaemia reactive cells. Allogeneic viral superantigen 7 reactive Vβ6+ T cells are able to transfer graft-vs-leukaemia reactivity and to kill specifically viral superantigen 7+ ESb-MP tumour cells in vitro. Here we elucidate the mechanism of this superantigen specific cell lysis. Already 10 min after co-incubation with in vitro stimulated Vβ6+ T cells, viral superantigen 7+ ESb-MP tumour cells show an apoptotic phenotype (Annexin V-positivity, DNA-fragmentation). This extremely rapid type of cell death is not mediated by the death inducing ligands CD95L, TRAIL and TNF but by perforin and granzyme B. Surprisingly, neither mitochondria nor any of the known caspases appear to be involved in this type of tumour cell killing. In contrast, nitric oxide, released by activated macrophages and endothelial cells, induces in the same tumour cells another type of apoptosis which is much slower and involves mitochondria and caspase activation. A synergistic effect between the two different effector mechanisms of superantigen reactive donor cytotoxic T lymphocytes and nitric oxide releasing host macrophages and endothelial cells might explain the effective immune rejection of even advanced metastasised cancer in this graft-vs-leukaemia animal model

Fas-Mediated Apoptosis Regulates the Composition of Peripheral αβ T Cell Repertoire by Constitutively Purging Out Double Negative T Cells

BACKGROUND: The Fas pathway is a major regulator of T cell homeostasis, however, the T cell population that is controlled by the Fas pathway in vivo is poorly defined. Although CD4 and CD8 single positive (SP) T cells are the two major T cell subsets in the periphery of wild type mice, the repertoire of mice bearing loss-of-function mutation in either Fas (lpr mice) or Fas ligand (gld mice) is predominated by CD4(-)CD8(-) double negative alphabeta T cells that also express B220 and generally referred to as B220+DN T cells. Despite extensive analysis, the basis of B220+DN T cell lymphoproliferation remains poorly understood. In this study we re-examined the issue of why T cell lymphoproliferation caused by gld mutation is predominated by B220+DN T cells. METHODOLOGY AND PRINCIPAL FINDINGS: We combined the following approaches to study this question: Gene transcript profiling, BrdU labeling, and apoptosis assays. Our results show that B220+DN T cells are proliferating and dying at exceptionally high rates than SP T cells in the steady state. The high proliferation rate is restricted to B220+DN T cells found in the gut epithelium whereas the high apoptosis rate occurred both in the gut epithelium and periphery. However, only in the periphery, apoptosis of B220+DN T cell is Fas-dependent. When the Fas pathway is genetically impaired, apoptosis of peripheral B220+DN T cells was reduced to a baseline level similar to that of SP T cells. Under these conditions of normalized apoptosis, B220+DN T cells progressively accumulate in the periphery, eventually resulting in B220+DN T cell lymphoproliferation. CONCLUSIONS/SIGNIFICANCE: The Fas pathway plays a critical role in regulating the tissue distribution of DN T cells through targeting and elimination of DN T cells from the periphery in the steady state. The results provide new insight into pathogenesis of DN T cell lymphoproliferation

Using death to one's advantage: HIV modulation of apoptosis

Infection by human immunodeficiency virus (HIV) is associated with an early immune dysfunction and progressive destruction of CD4+ T lymphocytes. This progressive disappearance of T cells leads to a lack of immune control of HIV replication and to the development of immune deficiency resulting in the increased occurrence of opportunistic infections associated with acquired immune deficiency syndrome (AIDS). The HIV-induced, premature destruction of lymphocytes is associated with the continuous production of HIV viral proteins that modulate apoptotic pathways. The viral proteins, such as Tat, Env, and Nef, are associated with chronic immune activation and the continuous induction of apoptotic factors. Viral protein expression predisposes lymphocytes, particularly CD4+ T cells, CD8+ T cells, and antigen-presenting cells, to evolve into effectors of apoptosis and as a result, to lead to the destruction of healthy, non-infected T cells. Tat and Nef, along with Vpu, can also protect HIV-infected cells from apoptosis by increasing anti-apoptotic proteins and down- regulating cell surface receptors recognized by immune system cells. This review will discuss the validity of the apoptosis hypothesis in HIV disease and the potential mechanism(s) that HIV proteins perform in the progressive T cell depletion observed in AIDS pathogenesis. Originally published Leukemia, Vol. 15, No. 3, Mar 200

Estudo biológico e comportamental de lagartas de Spodoptera frugiperda visando à produção de Baculovírus spodoptera

A utilização de bioinseticida a base de Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) possui potencial para o controle de Spodoptera frugiperda (Lepidoptera: Noctuidae), porém sua obtenção em larga escala depende da maximização da produção in vivo. Assim, alguns fatores biológicos e comportamentais devem ser estudados para aperfeiçoar a produção de SfMNPV com intuito de disponibilizar um bioinseticida eficiente, economicamente viável e que possa ser usado no manejo de S. frugiperda nos mais diversos sistemas agrícolas. Entre os fatores relacionados ao hospedeiro, a temperatura e a idade para inoculação do vírus são de extrema importância, pois interferem diretamente no ciclo de vida e na replicação viral. O comportamento também deve ser avaliado, para evitar condições de criação do hospedeiro que favoreçam o canibalismo e causa prejuízo na multiplicação in vivo do SfMNPV. Assim, objetivou-se determinar a melhor condição térmica para criar as lagartas e a idade ideal, para inocular e multiplicar o vírus no hospedeiro, bem como, verificar a ocorrência do comportamento canibal em lagartas de S. frugiperda. Os experimentos foram conduzidos no Laboratório de Controle Microbiano de Insetos do Núcleo de Desenvolvimento Científico e Tecnológico em Manejo Fitossanitário de Pragas e Doenças (NUDEMAFI), localizado no Centro de Ciências Agrárias da UFES, em Alegre, Espírito Santo, Brasil. A pesquisa foi desenvolvida em duas etapas, a primeira para determinar a condição térmica e a idade ideais para criar e inocular, respectivamente, o hospedeiro com o vírus, para multiplicação in vivo de SfMNPV. A segunda etapa foi para avaliar o comportamento canibal de lagartas da espécie S. frugiperda criadas a 22, 25 e 31°C, inoculadas com SfMNPV quando com idades de 10, 8 e 4 dias, respectivamente, e mantidas em diferentes densidades populacionais (5, 10, 25 e 50 lagartas por recipiente). A mortalidade diminuiu com o aumento da temperatura e da idade do hospedeiro nas temperaturas de 25, 28 e 31 °C. O aumento na taxa de canibalismo foi 12 diretamente proporcional à densidade populacional quando as lagartas foram criadas a 22 °C, inoculadas aos 10 dias de idade e 25 ºC, inoculadas aos 8 dias e atingiram 63,5 e 62,5%, respectivamente na densidade populacional de 50 lagartas. Mas, quando as lagartas foram criadas a 31ºC e inoculadas com idade de 4 dias, a densidade populacional não afetou o comportamento canibal, taxa média de 24%, inferior aos outros tratamentos com 50 lagartas por recipiente. Demonstrando que é viável para a multiplicação viral, criar lagartas a 31 °C e aos 4 dias de idade inocular o vírus, podendo a partir de então colocar até 50 lagartas por recipiente, o que reduz a mão-de-obra necessária para individualizar as lagartas e otimiza o espaço físico em uma biofábrica. Portanto, se para otimizar o processo produção viral e o serviço em uma biofábrica, é preciso maximizar a produção viral, reduzir o tempo de multiplicação do vírus e o canibalismo entre as lagartas, com ausência de contaminação da criação, a temperatura e idade ideais para criação massal de S. frugiperda e inoculação do vírus nas lagartas, respectivamente, visando produção de baculovírus em larga escala são de 31 ºC e 4 dias