Showing papers in "Immunology and Cell Biology in 1999"

Fluorescent dyes for lymphocyte migration and proliferation studies

Abstract: Fluorescent dyes are increasingly being exploited to track lymphocyte migration and proliferation. The present paper reviews the properties and performance of some 14 different fluorescent dyes that have been used during the last 20 years to monitor lymphocyte migration. Of the 14 dyes discussed, two stand out as being the most versatile in terms of long-term tracking of lymphocytes and their ability to quantify lymphocyte proliferation. They are the intracellular covalent coupling dye carboxyfluorescein diacetate succinimidyl ester (CFSE) and the membrane inserting dye PKH26. Both dyes have the advantage that they can be used to track cell division, both in vitro and in vivo, due to the progressive halving of the fluorescence intensity of the dyes in cells after each division. However, CFSE appears to have the edge over PKH26 based on homogeneity of lymphocyte staining and cost. Two other fluorescent dyes, although not suitable for lymphocyte proliferation studies, are valuable tracking dyes for short-term (up to 3 day) lymphocyte migration experiments, namely the DNA-binding dye Hoechst 33342 and the cytoplasmic dye calcein. In the future it is highly likely that additional fluorescent dyes, with different spectral properties to CFSE, will become available, as well as membrane inserting fluorescent dyes that more homogeneously label lymphocytes than PKH26.

Cell death induced by the Fas/Fas ligand pathway and its role in pathology.

Abstract: Engagement of the cell death surface receptor Fas by Fas ligand (FasL) results in apoptotic cell death, mediated by caspase activation. Cell death mediated via Fas/FasL interaction is important for homeostasis of cells in the immune system and for maintaining immune-privileged sites in the body. Killing via the Fas/FasL pathway also constitutes an important pathway of killing for cytotoxic T cells. Fas ligand is induced in activated T cells, resulting in activation-induced cell death by the Fas/FasL pathway. Recently it has been shown that the Fas receptor can also be up-regulated following a lesion to the cell, particularly that induced by DNA-damaging agents. This can then result in killing of the cell by a Fas/FasL-dependent pathway. Up-regulation of Fas receptor following DNA damage appears to be p53 dependent.

The role of CTLA-4 in the regulation of T cell immune responses.

Abstract: Over the past few years a great deal of research has examined how T cell-dependent immune responses are initiated and subsequently regulated. Ligation of the TCR with an antigenic peptide bound to an MHC protein on a professional APC provides the crucial antigen-specific stimulus required for T cell activation. Interaction of CD28 with CD80 or CD86 molecules on APC initiates a costimulatory or second signal within the T cell which augments and sustains T cell activation initiated through the TCR. However, recently it has become clear that T cell immune responses are a result of a balance between stimulatory and inhibitory signals. Cytotoxic T lymphocyte-associated molecule-4 (CTLA-4) is a cell surface molecule that is expressed nearly exclusively on CD4+ and CD8+ T cells. Investigation into the role of CTLA-4 in the regulation of T cell immune responses has revealed that CTLA-4 is a very important molecule involved in the maintenance of T cell homeostasis. In the present review, evidence for the proposed inhibitory role of CTLA-4 is examined and a model suggesting a role for CTLA-4 in both early and late stages of T cell activation is presented.

The IGF axis and programmed cell death.

Abstract: Insulin-like growth factors (IGF) are mitogenic peptides that have been implicated as positive regulators of cellular proliferation. In recent years, several studies have suggested an additional role for the IGF axis in the regulation of apoptosis. Signalling through the IGF receptor has been shown to have a potent survival function and protect cells from a variety of apoptotic stimuli. The actions of IGF are regulated by a family of high-affinity IGF binding proteins (IGFBP), which sequester the IGF from the IGF receptor. However, there is some evidence that one of these binding proteins, IGFBP-3, may have its own pro-apoptotic effects that are independent of its ability to modulate IGF bioavailability. In addition, it has been suggested that the tumour suppressor p53, a crucial mediator of apoptosis in response to cellular stress, may elicit several of its apoptotic effects through manipulation of components of the IGF axis. This review summarizes what is currently known about the role of the IGF system in the regulation of apoptosis, highlighting its implications in the context of tumorigenesis.

The role of B7 costimulation in T-cell immunity

Abstract: CD4+ T cells are considered to be the major controlling element of the adaptive immune response. They recognize foreign peptides by interaction of the T cell receptor (TCR) with peptide complexed to major histocompatibility complex (MHC) class II molecules on the surface of antigen presenting cells (APC). Once activated, CD4+ T cells orchestrate the various phases of the immune response. They are responsible for the production of numerous cytokines, which activate specific immune effector cell populations including B cells, eosinophils, mast cells and macrophages. Not surprisingly, the activation of CD4+ T cells needs to be tightly regulated and is subject to finely tuned control mechanisms. The requirement for a second or 'costimulatory' signal, in addition to the antigenic signal, provides a key element for the exquisite control of T cell activation. One of the major signalling pathways responsible for delivery of this costimulatory signal is induced by interaction of CD28 on T cells with B7 molecules found only on APC. The present review outlines our current understanding of the physiological role of B7 costimulatory signals in regulating CD4+ T cell responses.

Regulation of caspase activation and apoptosis by cellular zinc fluxes and zinc deprivation: A review.

Abstract: Regulation of caspase activation and apoptosis by cellular zinc fluxes and zinc deprivation: A review

Divided we stand: Tracking cell proliferation with carboxyfluorescein diacetate succinimidyl ester

Abstract: Most techniques for assessing cell division can either detect limited numbers of cell divisions (bromodeoxyuridine incorporation) or only quantify overall proliferation (tritiated thymidine incorporation). In the majority of cases, viable cells of known division history cannot subsequently be obtained for functional studies. The cells of the immune system undergo marked proliferation and differentiation during the course of an immune response. The relative lack of an organized structure of the lymphohaemopoietic system, in contrast with other organ systems, makes lineage interrelationships difficult to study. Coupled with the remarkable degree of mobility engendered by recirculation, the differentiation occurring along with cell division in the immune system has not been readily accessible for investigation. The present article reviews the development of a cell division analysis procedure based on the quantitative serial halving of the membrane permeant, stably incorporating fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE or CFDA, SE). The technique can be used both in vitro and in vivo, allowing eight to 10 successive divisions to be resolved by flow cytometry. Furthermore, viable cells from defined generation numbers can be sorted by flow cytometry for functional analysis.

Involvement of ribosomal proteins in regulating cell growth and apoptosis: translational modulation or recruitment for extraribosomal activity?

Abstract: Gene recruitment is a mechanism of molecular evolution whereby a gene product can function in more than one distinct capacity The 'one gene-dual function' phenomenon is well illustrated by crystallins, structural proteins that play both specialized roles in the eye lens and also 'housekeeping' enzyme roles Ribosomal proteins are integral components of the basal cellular machinery involved in protein synthesis, whose roles have been regarded collectively as important, but individually somewhat mundane However, various individual ribosomal proteins and also translation initiation and elongation factors have been found to play roles in regulating cell growth, transformation and death, giving rise to increasing speculation that components of the translational apparatus can act as multifunctional proteins Recently, we have shown that ribosomal protein S3a (RPS3a) plays important roles in cell transformation and death, whereby constitutively or transiently enhanced RPS3a expression can be regarded as 'priming' a cell for apoptosis and suppression of such enhanced expression as 'execution' While it is unclear whether RPS3a acts in a capacity mechanistically distinct from that in translation, such a possibility is discussed in this article in the light of recent, although not exhaustively reviewed, findings implicating the involvement of other individual ribosomal proteins in modulating and/or effecting changes in cellular responses and growth patterns in an extraribosomal capacity independent of their conventional role in translation

Quantitative analysis of lymphocyte differentiation and proliferation in vitro using carboxyfluorescein diacetate succinimidyl ester.

Abstract: Mature T and B lymphocytes respond to receptor-delivered signals received during and following activation. These signals regulate the rates of cell death, growth, differentiation and migration that ultimately establish the behaviour patterns collectively referred to as immune regulation. We have been pursuing the philosophy that in vitro systems of lymphocyte stimulation, when analysed quantitatively, help reveal the logical attributes of lymphocyte behaviour. The development of carboxyfluorescein diacetate succinimidyl ester (CFSE) to track division has enabled the variable of division number to be incorporated into these quantitative analyses. Our studies with CFSE have established a fundamental link between differentiation and division number. Isotype switching, expression of T cell cytokines, surface receptor alterations and changes to intracellular signalling components all display independent patterns of change with division number. The stochastic aspects of these changes and the ability of external signals to independently regulate them argue for a probabilistic modelling framework for describing and understanding immune regulation.

Osteoprotegerin ligand: a common link between osteoclastogenesis, lymph node formation and lymphocyte development.

Abstract: The TNF-family molecule osteoprotegerin ligand (OPGL; also known as TRANCE, RANKL or ODF) has been identified as the osteoclast differentiation factor and a regulator of T cell-dendritic cell interactions in the immune system. Surprisingly, the same molecule was identified as a crucial factor in early lymphocyte development and lymph node organogenesis. We will discuss the role of OPGL in bone remodelling and the immune system.

Molecular mechanisms of ionizing radiation-induced apoptosis.

Abstract: Ionizing radiation activates not only signalling pathways in the nucleus as a result of DNA damage, but also signalling pathways initiated at the level of the plasma membrane. Proteins involved in DNA damage recognition include poly(ADP ribose) polymerase (PARP), DNA-dependent protein kinase, p53 and ataxia-telangiectasia mutated (ATM). Many of these proteins are inactivated by caspases during the execution phase of apoptosis. Signalling pathways outside the nucleus involve tyrosine kinases such as stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), protein kinase C, ceramide and reactive oxygen species. Recent evidence shows that tumour cells resistant to ionizing radiation-induced apoptosis have defective ceramide signalling. How these signalling pathways converge to activate the caspases is presently unknown, although in some cell types a role for calpain has been suggested.

The effect of molecular weight and beta-1,6-linkages on priming of macrophage function in mice by (1,3)-beta-D-glucan.

Abstract: The effect of molecular weight and β-1,6-linkages on priming of macrophage function in mice by (1,3)-β- D -glucan

Chemokines and chemokine receptors in infectious diseases

Abstract: Today, 10 years after the discovery of IL-8, chemokines (chemotactic cytokines) are seen as the stimuli that largely control leucocyte migration. Chemokines are low molecular weight chemoattractant cytokines secreted by a variety of cells, including leucocytes, epithelial cells, endothelial cells, fibroblasts and numerous other cell types. They are produced in response to exogenous stimuli, such as viruses and bacterial LPS, and endogenous stimuli, such as IL-1, TNF and IFN. These factors mediate chemotaxis and leucocyte activation. They also regulate leucocyte extravasation from the blood and/or lymph vessel luminal surface to the tissue space, the site of inflammation. There is no doubt that chemokines and chemokine receptors are critical for defence against infectious pathogens. It is also clear that these pathogens have evolved to accommodate the workings of the host immune system. Survival of these infectious agents appears dependent upon strategies that can evade, suppress, counteract or otherwise confound the constellation of host responses to invading pathogens. In this regard, the chemokines and their receptors are a major target. Reviewed in the present paper are several examples in which microbial pathogens have usurped the mammalian chemokine system to subvert the host immune response.

Carboxyfluorescein succinimidyl ester-based proliferative assays for assessment of T cell function in the diagnostic laboratory.

Abstract: Immune deficiency diseases are often accompanied by abnormalities in one or both arms of the specific immune system. Impairment can often be detected as a decrease in the number of T or B lymphocytes or their products in the circulation, but questions are often asked as to the functional capabilities of T lymphocytes in patients with recurrent infections. Function of T cells has traditionally been measured by their uptake of [3H]- thymidine following stimulation with antigen or mitogen in vitro. However, the ability of carboxyfluorescein succinimidyl ester (CFSE) to label lymphocytes intracellularly and track their mitotic activity by progressive two-fold reduction in fluorescence intensity prompted an alternative methodology based on flow cytometry, an approach which has the advantage of allowing specific gating on particular T cell subsets and simultaneous assessment of activation markers. This method was therefore evaluated for T cell responses to mitogen and antigen. Phytohaemagglutinin-induced blast transformation of CFSE-labelled T cells was reflected by an increase in forward and orthogonal light scatter and a progressive two-fold decrease in CFSE fluorescence intensity. These changes allowed the derivation of various measures of mitotic activity, which correlated well with [3H]-thymidine uptake. Patients with T cell functional deficiencies showed impairment in their responses by both assays, whereas the CFSE-based assay demonstrated that impaired blastogenesis was not simply due to depressed T cell numbers. Concomitant measurement of the activation markers CD69 and CD25 showed that CD69 was rapidly expressed on non-mitotic cells and that this expression was progressively diluted with subsequent rounds of cell division. In contrast, CD25 expression was unaffected by cell cycle, but was expressed in proportion to the PHA dose. Antigen-specific responsiveness to Candida was also assessed using a CFSE-based assay. Initial gating on the relatively minor population of T cells that underwent blast transformation demonstrated progressive twofold dilutions of CFSE intensity in responsive cells. These normal Candida responses, found in patients who had recovered from Candida infection, contrasted with those who had not been infected with Candida or who had chronic recurrent infection, in whom neither blast transformation nor significant mitosis could be detected. Again, there was good correlation with [3H]-thymidine uptake. The CFSE-based assays are equivalent to traditional measures of mitogen- and antigen-specific T cell responsiveness in the diagnostic laboratory and have significant advantages in terms of decreased labour intensiveness, avoidance of radioactivity, the ability to gate on a specific population of lymphocytes and the concomitant measurement of activation markers.

Mucosa-associated lymphoid tissues as sites for uptake, carriage and excretion of tubercle bacilli and other pathogenic mycobacteria.

Abstract: Pathogenic mycobacteria, including those that cause tuberculosis and paratuberculosis, cross mucosal barriers by endocytosis within mucosal lymphoepithelial sites. These entry sites commonly include oropharyngeal and nasopharyngeal tonsils and Peyer's patches. Bacilli discharged at the basolateral surfaces of engulfing epithelial M cells are taken up by professional antigen-presenting cells associated with T lymphocytes of the parafollicular area. Dendritic cells and macrophages in these sites allow mycobacterial replication, due to the permissive immunological environment in lymphoepithelial tissues. Abrogation of local delayed-type hypersensitivity reactions generally ensures continuing integrity and function of these tissues. Phagocytes containing intracellular mycobacteria disseminate infection to other parts of the body and also probably migrate back onto the mucosal surface to shed bacilli.

Signalling by CD95 and TNF receptors: not only life and death

Abstract: Members of the TNF family of receptors play important roles in normal physiology and in defence. The recent rapid progress in the understanding of the mechanisms of apoptosis has been accompanied by assumptions that TNF family receptors such as CD95(Fas/APO-1) only have a role in regulating cell survival. While regulation of cell death is one important function of TNF family receptors, they are capable of activating signal transduction pathways that have many other effects. The present review will focus on signalling of some TNF family receptors in the immune system, not only for apoptosis, but also for survival or activation.

Salivary IgA subclasses and infection risk in elite swimmers.

Abstract: The concentrations of total IgA, IgA1 and IgA2 were measured in saliva collected from 25 elite swimmers in the early and late phases of a 7 month training season and compared with the number of respiratory infections during the season. The IgA1 concentrations in the early phase of the training season were significantly associated (P = 0.01) with the number of respiratory infection episodes during the training season. The lower the concentration of IgA1, the greater the number of infection episodes. Swimmers with four or more infections during the training season had significantly lower salivary IgA1 concentrations than those with less than four infection episodes (P = 0.01). The proportion of IgA1 in the saliva of the elite swimmers (80%) was higher than for normal non-exercising adults (60%). A small proportion of athletes had salivary IgA2 concentrations below the detection limit of the assay and the mean concentration of IgA2 was significantly lower than the concentrations for a normal adult population (P = 0.01). This study suggests that measurement of IgA subclasses, in particular IgA1, at the commencement of a training season may predict infection risk in elite swimmers.

NK cells and apoptosis.

Abstract: Natural killer (NK) cells are a cell of the innate immune system that play an important role in the early response to viral infections and tumours. Natural killer cells are cytolytic, and secrete cytokines that influence the developing antigen-specific immune response. In the present article the NK cell surface molecules regulating effector function, the NK cell effector mechanisms involved in apoptosis, and the role of NK cell effector mechanisms in immune responses are reviewed.

Dendritic cells: The driving force behind autoimmunity in rheumatoid arthritis?

Abstract: Dendritic cells (DC) are likely to play a significant role in immune-mediated diseases such as autoimmunity and allergy To date there are few treatments capable of inducing permanent remission in rheumatoid arthritis (RA) and elucidation of the role of DC may provide specific strategies for disease intervention Dendritic cells have proven to be powerful tools for immunotherapy and investigations are under way to determine their clinical efficacy in transplantation and viral and tumour immunotherapy The present review will focus on the current view of DC and their role in autoimmunity, in particular RA Two possible roles for DC in the pathogenesis of RA will be proposed, based on recent advances in the field

Dendritic cells at the end of the Millennium

Abstract: We have recently proposed a dual role for dendritic cells (DC) in the amplification of innate immune responses and in the activation of adaptive immune responses. The DC are localized along the major routes of entry of micro-organisms, where they perform a sentinel function for incoming pathogens. Soon after interaction with appropriate stimuli, DC undergo a coordinated process of maturation and respond to danger signals by re- programming their functions. The DC first regulate leucocyte recruitment at the site of inflammation, through the production of chemokines, inflammatory cytokines and interferons, and then they acquire migratory properties and undergo a rapid switch in chemokine receptor expression. This allows them to leave the inflamed tissue and to reach the lymph node T cell area. During this migration, DC complete their maturation process and acquire the ability to prime T cell responses. Thus, DC bridge innate and adaptive immunity.

Activation-Induced Apoptosis of Autoreactive and Alloreactive T Lymphocytes in the Target Organ as a Major Mechanism of Tolerance

Abstract: Normal individuals have mature T lymphocytes that are capable of reacting to self-antigens and can be activated by cross-reacting environmental antigens. The mechanism that maintains immune tolerance and prevents these activated autoreactive T cells from causing autoimmune disease is unclear. We have previously hypothesized that activation-induced apoptosis of previously activated autoreactive T cells in the target organ is a major mechanism for maintaining tolerance. Here I review the current evidence to support this hypothesis. It is proposed that when activated autoreactive T cells enter the target organ, they are reactivated mainly by non-professional antigen-presenting cells (APC) and deleted by activation-induced apoptosis through the Fas (CD95) pathway before producing significant target organ damage. This apoptosis occurs because the reactivated T cells do not receive sufficient costimulation from the non-professional APC to up-regulate their expression of Bcl-2-related anti-apoptotic proteins, which inhibit the CD95 pro-apoptotic pathway. This is in contrast to the situation in peripheral lymphoid organs, where reactivation of T cells by professional APC results in sufficient costimulation-induced up-regulation of Bcl-2-related proteins to inhibit the CD95 pathway and allow T cell proliferation and survival as memory T cells. Activation-induced apoptosis of alloreactive T cells in allografts can similarly account for spontaneous allograft acceptance, as occurs after MHC-mismatched liver transplantation.

Modulation of immune function by a modified bovine whey protein concentrate

Abstract: The commercial preparation of dairy foodstuffs generates large volumes of by-products, many of which have as yet undocumented effects on mammalian immune function. In the present report, a modified whey protein concentrate (mWPC), derived as a by-product from the commercial manufacture of cheese, was tested for its ability to modulate murine immune function in vitro. The mWPC suppressed T and B lymphocyte proliferative responses to mitogens in a dose-dependent fashion. The mWPC also suppressed alloantigen-induced lymphocyte proliferation during a mixed leucocyte reaction, but showed no suppressive effect against IL-2-sustained proliferation of mitogen-activated T cell blasts. Other indices of lymphocyte activation, such as cytokine secretion and the formation of activated (CD25+) T cell blasts, were suppressed by the mWPC, suggesting that the mode of suppression may be to inhibit the lymphocyte activation process. Enzymatic digestion by pepsin and pancreatin, under physiologically realistic conditions in vitro, ablated the immunomodulatory function of the mWPC. These results are discussed in relation to the potential development of complex-mixture dairy products into health-modulating products.

Carboxyfluorescein diacetate succinimidyl ester and the virgin lymphocyte: a marriage made in heaven.

Abstract: Carboxyfluorescein diacetate succinimidyl ester (CFSE) labelling of naive lymphocyte populations provides unique insights into the immune response. The clonal nature of immune responses, necessitating clonal expansion to achieve a sufficiently large number of Ag-reactive effector cells, combined with the dependence of lymphocyte differentiation on cell division, underlie the usefulness of CFSE in understanding the factors that regulate responses both in vitro and in vivo. We have combined CFSE labelling with Ag receptor transgenic models, using seven channel flow cytometry to track the correlation between cell division and a number of other parameters, such as surface expression of activation markers, cytokine receptors and homing receptors, cytokine production, cytotoxic activity and indicators of apoptosis. Our data have allowed us to classify and understand immune responses in novel ways, suggesting many further avenues of enquiry and indicating previously unrecognized relationships between cell division and eventual cell fate.

Regulation of pro-apoptotic leucocyte granule serine proteinases by intracellular serpins.

Abstract: Top of pageAbstract Caspase activation and apoptosis can be initiated by the introduction of serine proteinases into the cytoplasm of a cell. Cytotoxic lymphocytes have evolved at least one serine proteinase with specific pro-apoptotic activity (granzyme B), as well as the mechanisms to deliver it into a target cell, and recent evidence suggests that other leucocyte granule proteinases may also have the capacity to kill if released into the interior of cells. For example, the monocyte/granulocyte proteinase cathepsin G can activate caspases in vitro, and will induce apoptosis if its entry into cells is mediated by a bacterial pore-forming protein. The potent pro-apoptotic activity of granzyme B and cathepsin G suggests that cells producing these (or other) proteinases would be at risk from self-induced death if the systems involved in packaging, degranulation or targeting fail and allow proteinases to enter the host cell cytoplasm. The purpose of the present review is to describe recent work on a group of intracellular serine proteinase inhibitors (serpins) which may function in leucocytes to prevent autolysis induced by the granule serine proteinases. Keywords: apoptosis, cathepsin G, cytotoxicity, granule, granzyme B, leucocyte, M/NEI, phagocytosis, PI-6, PI-9, serpin

Analysis of growth kinetics by division tracking

Abstract: Cell division tracking using fluorescent dyes, such as carboxyfluorescein diacetate succinimidyl ester, provides a unique opportunity for analysis of cell growth kinetics. The present review article presents new methods for enhancing resolution of division tracking data as well as derivation of quantities that characterize growth from time-series data. These include the average time between successive divisions, the proportion of cells that survive and the proliferation per division. The physical significance of these measured quantities is interpreted by formulation of a two-compartment model of cell cycle transit characterized by stochastic and deterministic cell residence times, respectively. The model confirmed that survival is directly related to the proportion of cells that enter the next cell generation. The proportion of time that cells reside in the stochastic compartment is directly related to the proliferation per generation. This form of analysis provides a starting point for more sophisticated physical and biochemical models of cell cycle regulation.

Does HIV cause depletion of CD4+ T cells in vivo by the induction of apoptosis?

Abstract: The central pathogenic feature of AIDS is the dramatic loss of CD4+ lymphocytes. Despite more than a decade of intense research, the exact mechanism by which HIV causes this is still not understood. A major model for T cell depletion, proposed originally by Ameison and Capron in a report published in 1991, is that HIV sensitizes CD4+ T cells for activation-induced apoptosis. The apoptotic model of T cell depletion is discussed, and experiments that address the questions of whether apoptosis is restricted to infected cells or 'bystander' T cells, and whether T cell apoptosis requires participation of separate HIV-infected haematopoietic cell populations, are reviewed.

Targeted disruption of caspase genes in mice: What they tell us about the functions of individual caspases in apoptosis

Abstract: Cysteine proteases of the caspase family are crucial mediators of apoptosis. All mammalian cells contain a large number of caspases. Although many caspases are activated in a cell committed to apoptosis, recent data from caspase gene knockout mice suggest that individual caspases may be involved in the cell and stimulus-specific pathways of cell death. The gene disruption studies also establish the functional hierarchy between two structurally distinct classes of caspases. The present review discusses these recent findings and elaborates on how these mutant mouse models have helped the understanding of the mechanisms that govern programmed cell death in the immune and other systems.

Bcl-2 genes and growth factors in the pathology of ischaemic acute renal failure.

Abstract: For the past decade, an attempt has been made by many research groups to define the roles of the growing number of Bcl-2 gene family proteins in the apoptotic process. The Bcl-2 family consists of pro-apoptotic (or cell death) and anti-apoptotic (or cell survival) genes and it is the balance in expression between these gene lineages that may determine the death or survival of a cell. The majority of studies have analysed the role/s of the Bcl-2 genes in cancer development. Equally important is their role in normal tissue development, homeostasis and non-cancer disease states. Bcl-2 is crucial for normal development in the kidney, with a deficiency in Bcl-2 producing such malformation that renal failure and death result. As a corollary, its role in renal disease states in the adult has been sought. Ischaemia is one of the most common causes of both acute and chronic renal failure. The section of the kidney that is most susceptible to ischaemic damage is the outer zone of the outer medulla. Within this zone the proximal tubules are most sensitive and often die by necrosis or desquamate. In the distal nephron, apoptosis is the more common form of cell death. Recent results from our laboratory have indicated that ischaemia-induced acute renal failure is associated with up-regulation of two anti-apoptotic Bcl-2 proteins (Bcl-2 and Bcl-XL) in the damaged distal tubule and occasional up-regulation of Bax in the proximal tubule. The distal tubule is a known reservoir for several growth factors important to renal growth and repair, such as insulin-like growth factor-1 (IGF-1) and epidermal growth factor (EGF). One of the likely possibilities for the anti-cell death action of the Bcl-2 genes is that the protected distal cells may be able to produce growth factors that have a further reparative or protective role via an autocrine mechanism in the distal segment and a paracrine mechanism in the proximal cells. Both EGF and IGF-1 are also up-regulated in the surviving distal tubules and are detected in the surviving proximal tubules, where these growth factors are not usually synthesized. As a result, we have been using in vitro methods to test: (i) the relative sensitivities of renal distal and proximal epithelial cell populations to injury caused by mechanisms known to act in ischaemia-reperfusion; (ii) whether a Bcl-2 anti-apoptotic mechanism acts in these cells; and (iii) whether an autocrine and/or paracrine growth factor mechanism is initiated. The following review discusses the background to these studies as well as some of our preliminary results.

Anti-viral strategies of cytotoxic T lymphocytes are manifested through a variety of granule-bound pathways of apoptosis induction.

Abstract: Cytotoxic T cells and natural killer cells together constitute a major defence against virus infection, through their ability to induce apoptotic death in infected cells. These cytolytic lymphocytes kill their targets through two principal mechanisms, and one of these, granule exocytosis, is essential for an effective in vivo immune response against many viruses. In recent years, the authors and other investigators have identified several distinct mechanisms that can induce death in a targeted cell. In the present article, it is postulated that the reason for this redundancy of lethal mechanisms is to deal with the array of anti-apoptotic molecules elaborated by viruses to extend the life of infected cells. The fate of such a cell therefore reflects the balance of pro-apoptotic (immune) and anti-apoptotic (viral) strategies that have developed over eons of evolutionary time.

Physiological effects and adjuvanticity of recombinant brushtail possum TNF-alpha.

Abstract: The present paper describes the physiological properties of recombinant possum TNF-alpha and an adjuvant effect on antibody responses to the model protein antigen, keyhole limpet haemocyanin (KLH). For these studies recombinant possum TNF-alpha was produced in the yeast Pichia pastoris. The recombinant cytokine was secreted into the culture medium and purified by gel filtration. Possum TNF-alpha produced in this expression system was N-glycosylated and bioactive in two different assays. In a murine fibroblast L929 cytotoxicity assay, the possum TNF-alpha had lower specific activity compared to human TNF-alpha, while in a possum-specific assay, possum TNF-alpha enhanced the proliferation of PHA-stimulated possum thymocytes and was more active than human TNF-alpha. The physiological effect of the recombinant possum TNF-alpha was investigated in groups of possums administered doses of 6, 30 or 150 micrograms of cytokine. For each dose, TNF-alpha caused profound effects on the numbers of circulating leucocytes characterized by a three-to-four-fold increase in neutrophil numbers at 6-24 h after injection and an initial sharp decrease in lymphocyte numbers. The efficacy of TNF-alpha as an immunological adjuvant was determined in possums administered KLH (125 micrograms) in an aqueous or Al(OH)3-based formulation with or without added recombinant TNF-alpha (150 micrograms). Serum antibody responses to KLH were monitored by ELISA. The TNF-alpha stimulated two-fold and four-fold increases in antibody levels in aqueous and Al(OH)3-based vaccine formulations, respectively. The strongest antibody responses were observed in the group of possums that received KLH formulated in Al(OH)3 with addition of TNF-alpha.