proposed a set of recommendations for the definition of distinct cell death morphologies and for the appropriate use of cell death-related terminology, including 'apoptosis', 'necrosis' and 'mitotic catastrophe'. In view of the substantial progress in the biochemical and genetic exploration of cell death, time has come to switch from morphological to molecular definitions of cell death modalities. Here we propose a functional classification of cell death subroutines that applies to both in vitro and in vivo settings and includes extrinsic apoptosis, caspase-dependent or -independent intrinsic apoptosis, regulated necrosis, autophagic cell death and mitotic catastrophe. Moreover, we discuss the utility of expressions indicating additional cell death modalities. On the basis of the new, revised NCCD classification, cell death subroutines are defined by a series of precise, measurable biochemical features.

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Additional complexity in p73: induction by mitogens in lymphoid cells and identification of two new splicing variants e and z Dear Editor, p53 is a sequence specific transcription factor which transactivates several genes important in the apoptotic pathway, such as p21, mdm2, gadd45, bax and caspases. 1,2 Lack of apoptosis, with inappropriate cell proliferation in cancer is correlated with a high frequency of p53 mutations, in some tumours reaching 50%. Perhaps surprisingly, in view of the importance of p53 in regulating cell death and its strong phylogenetic conservation, only recently have homologous genes been identified.These (p63 and p73) show up to 63% aminoacid identity with p53 in the DNA binding, oligomerization and transcription activation domains, suggesting a similar mechanism of action to p53. 3±7 p63 is expressed as six different forms. 4 These use one of the two alternative transcription initiation sites, each transcript then being expressed as one of three alternatively spliced variants (a, b, g). Since the transcripts using the downstream ATG lack the first three exons coding for the transactivation domain, they act as natural dominant negative mutants of full length p63 and of p53. 4 The p73 gene comprises 14 exons and we have shown previously that in addition to the full length a form and the alternatively spliced transcript lacking exon 13 (b) other splice variants, lacking exon 11 (g) and exons 11, 12 and 13 (d) are also produced. 8 Stimulation of the T lymphoblastoid cell line, Jurkat, and human peripheral blood lymphocytes (PBL) with phytoemagglutinin (PHA) causes a 3 and 2.6-fold increase respectively in p73 expression by Northern blotting after 24 h (panel A). This is associated with induction of 34.7% and 21.2% of apoptotic cells respectively. No p53 was detected in Jurkat cells after PHA treatment (not shown).In order to discriminate the differential induction of the four p73 isoforms we performed an RT±PCR using isoformspecific primers on RNA extracted from cells treated under the same conditions. Panel B shows upregulation of a, b, g and d in PHA-treated PBL and Jurkat cells. Densitometric comparison of these with the housekeeping gene GAPDH showed that the increase in expression of each isoform were comparable (not shown).In addition, a new isoform was amplified from normal PBL. Cloning and sequencing of this p73e identified it as a splicing variant lacking exons 11 and 13 (panel C). To confirm the existence of p73e, we screened a panel of normal and tumour cell lines. As also shown in panel B, p73e was also present in the human hepatoma line HepG2, and a sixth isoform z was identified in the MCF7 human breast cancer cell line and in a human skin biopsy. p73 z is a further splice variant which lacks exons 11 and 12, and results in the loss of 96 aminoacids, the sequence continuing with the C-terminus of the a form (panel C). In p73e, loss of exon 11 deletes 50 aminoacids with a frame shift to the reading frame of the g isoform; splicing of exon 13 deletes an additional 31 aminoacids...

Acute viral respiratory infections may induce clinically relevant biological and physiological changes within the lower airway in susceptible individuals [1]. Although such infections are a potent trigger of asthma symptoms, there is as yet no consensus as to whether they can initiate asthma de novo [2]. Eighty per cent of asthma exacerbations in school-aged children and half of all adult asthma exacerbations have been associated with viral upper respiratory infections, mostly due to human rhinovirus (HRV) [3,4]. Studies on experimentally induced HRV infections suggest that the rhinovirus may induce a greater effect on lower airway inflammation and function in subjects with established asthma or allergy [5][6][7][8]. Separate observations indicate that atopic individuals may be more susceptible to the development of HRV infections [9]. The mechanisms of these events, however, have not been systematically investigated.The epithelial cells (EC) are the primary target of HRV infection in human airways. EC express on their surface the intercellular adhesion molecule (ICAM)-1, which is the site of attachment for 90% of the approximately 100 HRV serotypes [10][11][12]. ICAM-1 interacts physiologically with leukocyte function-associated antigen (LFA)-1, expressed on leukocytes, and thus plays a vital role in the recruitment and migration of immune effector cells to sites of local inflammation. Recent reports suggest that HRV may interfere with this ICAM-1/LFA-1 binding on leukocytes and thus disrupt immune responses that are dependent on this interaction. These effects could lead to a disorder of local airway immunity with increased risk of productive viral replication [13]. Furthermore, HRV per se influences the expression of ICAM-1 on EC. Studies by the present authors [14] and others [15,16] have shown that HRV significantly upregulate EC ICAM-1 expression, an effect that would facilitate viral cell attachment and entry.HRV infection induces the local production of cytokines, known to mediate the acute-phase reactions of airway inflammation [15,17,18]. These cytokines [19][20][21][22][23] can increase the expression of ICAM-1, although the level of induced expression depends on the specific mediator and the cell type [14,[24][25][26]. Recently, it has been shown that tumour necrosis factor (TNF)-α, interleukin (IL)-1β and IL-8 maintain their induced increase in cell ICAM-1 expression during HRV infection; in contrast, interferon (IFN)-γ increases ICAM-1 expression in uninfected cells but produces a persistent downregulation of ICAM-1 in These data suggest that the effects of Th2-associated cytokines on intercellular adhesion molecule-1 expression and recovery of infectious virus are dominant over the effects of the Th1-associated cytokines such as interferon-γ. Since the airway mucosa in atopic asthma is predominantly infiltrated by Th2 lymphocytes, these results could explain both the increased susceptibility to human rhinovirus infection in asthmatic patients and the associated exacerbation of asthma symptoms. Eur...

This study was designed to investigate the ability of bronchoalveolar and blood mononuclear cells to produce inflammatory mediators in vitro in pulmonary sarcoidosis. Seventeen patients with pulmonary sarcoidosis (stage I n=8; stage II/III n=9) and 10 normal controls were investigated. Bronchoalveolar and peripheral blood mononuclear cells were cultured in serum-free medium, without stimulant, for 24 h, and the supernatants analysed for concentrations of interleukin (IL)-1β (IL-1β), IL-2, IL-6, tumour necrosis factor-α (TNF-α), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ) and neopterin.Bronchoalveolar lavage cells (BALC) of sarcoid patients released significantly higher amounts of TNF-α, IL-6, IFN-γ and neopterin in comparison to normal controls. When smokers were excluded, there was also an increased release of IL-1β and GM-CSF. In the sarcoid group, the levels of IL-1β, IL-6, TNF-α and GM-CSF showed highly significant correlations between each other, but not with IL-2, IFN-γ or neopterin. Sarcoid patients whose BALC released more TNF-α or GM-CSF had higher percentage counts of alveolar macrophages but fewer lavage lymphocytes. In sarcoid patients, peripheral blood mononuclear cells (PBMNC) also released higher amounts of IL-1β, TNF-α, IL-6 and GM-CSF but less neopterin than normal controls. Patients whose PBMNC produced more IL-1β, IL-6 and GM-CSF had higher absolute and relative lavage neutrophil counts. No relationships were observed between cytokine release and radiographic or physiological markers of disease severity.We conclude from this study that sarcoid inflammation is associated with an increased and concerted release of monocyte/macrophage-derived cytokines not only in the lung but also in the peripheral blood. We speculate that the lymphokines, IFN-γ and IL-2, are not the primary triggers.