Local amplification of glucocorticoids by 11 beta-hydroxysteroid dehydrogenase type 1 promotes macrophage phagocytosis of apoptotic leukocytes

Glucocorticoids promote macrophage phagocytosis of leukocytes undergoing apoptosis. Prereceptor metabolism of glucocorticoids by 11beta-hydroxysteroid dehydrogenases (11beta-HSDs) modulates cellular steroid action. 11beta-HSD type 1 amplifies intracellular levels of active glucocorticoids in mice by reactivating corticosterone from inert 11-dehydrocorticosterone in cells expressing the enzyme. In this study we describe the rapid (within 3 h) induction of 11beta-HSD activity in cells elicited in the peritoneum by a single thioglycolate injection in mice. Levels remained high in peritoneal cells until resolution. In vitro experiments on mouse macrophages demonstrated that treatment with inert 11-dehydrocorticosterone for 24 h increased phagocytosis of apoptotic neutrophils to the same extent as corticosterone. This effect was dependent upon 11beta-HSD1, as 11beta-HSD1 mRNA, but not 11beta-HSD2 mRNA, was expressed in these cells; 11-dehydrocorticosterone was ineffective in promoting phagocytosis by Hsd11b1(-/-) macrophages, and carbenoxolone, an 11beta-HSD inhibitor, prevented the increase in phagocytosis elicited in wild-type macrophages by 11-dehydrocorticosterone. Importantly, as experimental peritonitis progressed, clearance of apoptotic neutrophils was delayed in Hsd11b1(-/-) mice. These data point to an early role for 11beta-HSD1 in promoting the rapid clearance of apoptotic cells during the resolution of inflammation and indicate a novel target for therapy.     

Quantitative Analysis of Immune Response and Erythropoiesis during Rodent Malarial Infection

Malarial infection is associated with complex immune and erythropoietic responses in the host. A quantitative understanding of these processes is essential to help inform malaria therapy and for the design of effective vaccines. In this study, we use a statistical model-fitting approach to investigate the immune and erythropoietic responses in Plasmodium chabaudi infections of mice. Three mouse phenotypes (wildtype, T-cell-deficient nude mice, and nude mice reconstituted with T-cells taken from wildtype mice) were infected with one of two parasite clones (AS or AJ). Under a Bayesian framework, we use an adaptive population-based Markov chain Monte Carlo method and fit a set of dynamical models to observed data on parasite and red blood cell (RBC) densities. Model fits are compared using Bayes'' factors and parameter estimates obtained. We consider three independent immune mechanisms: clearance of parasitised RBCs (pRBC), clearance of unparasitised RBCs (uRBC), and clearance of parasites that burst from RBCs (merozoites). Our results suggest that the immune response of wildtype mice is associated with less destruction of uRBCs, compared to the immune response of nude mice. There is a greater degree of synchronisation between pRBC and uRBC clearance than between either mechanism and merozoite clearance. In all three mouse phenotypes, control of the peak of parasite density is associated with pRBC clearance. In wildtype mice and AS-infected nude mice, control of the peak is also associated with uRBC clearance. Our results suggest that uRBC clearance, rather than RBC infection, is the major determinant of RBC dynamics from approximately day 12 post-innoculation. During the first 2–3 weeks of blood-stage infection, immune-mediated clearance of pRBCs and uRBCs appears to have a much stronger effect than immune-mediated merozoite clearance. Upregulation of erythropoiesis is dependent on mouse phenotype and is greater in wildtype and reconstitited mice. Our study highlights the informative power of statistically rigorous model-fitting techniques in elucidating biological systems.     

A theoretical study of random segregation of minicircles in trypanosomatids

The kinetoplast (k) DNA network of trypanosomatids is made up of approximately 50 maxicircles and the order of 10(4) minicircles. It has been proposed, based on various observations and experiments, that the minicircles are randomly segregated between daughter cells when the parent cell divides. In this paper, this random segregation hypothesis is theoretically tested in a population dynamics model to see if it can account for the observed phenomena. The hypothesis is shown to successfully explain, in Leishmania tarentolae, the observation that there are a few major and many minor minicircle classes, the fluctuations of minicircle class copy numbers over time, the loss of non-essential minicircle classes, the long survival times of a few of these classes and that these classes are likely to be the major classes within the population. Implications of the model are examined for trypanosomatids in general, leading to several predictions. The model predicts variation in network size within a population, variation in the average network size and large-scale changes in class copy number over long time-scales, an evolutionary pressure towards larger network sizes, the selective advantage of non-random over random segregation, very strong selection for the amplified class in Crithidia fasciculata if its minicircles undergo random segregation and that Trypanosoma brucei may use sexual reproduction to maintain its viability.     

Mathematical models of hierarchically structured cell populations under equilibrium with application to the epidermis

Abstract. There are three categories of keratinocytes in the germinative compartment of the epidermis – stem, transit-amplifying and post-mitotic. Their population structure is hierarchical. This means that stem cells differentiate into transit-amplifying cells which, after a few rounds of division, become post-mitotic cells. The cell processes of birth, differentiation, death and migration affect the composition and proliferation rate of the germinative compartment. These phenomena are quantified by various cell kinetic parameters. In this paper we derive equations that relate these parameters for different models of hierarchically structured cell populations in equilibrium. We include in the models asymmetric and symmetric division, variations in cell-cycle times, apoptosis and variation in the number of transit generations. We conclude that variation in cell-cycle times need only be considered if apoptosis is not negligible. If it is negligible, then only average cell-cycle times are needed. Unfortunately, it is impossible to predict the importance of apoptosis from the available experimental data. However, the strength of its effect is determined by the other parameters, especially the fraction of cycling stem cells. We show that variation in the number of transit generations can have a potentially large effect on cell birth rate. We also show that cell birth rate does not directly depend on the mean transit-amplifying cell-cycle time, only on the mean stem cell-cycle time. We argue that 'homogeneous cell population' equations should not be used to study hierarchical cell populations as has been done in the past. Finally we argue that stem cell parameters and transit-amplifying cell parameters should not be lumped together.     

The detection of Bifidobacterium adolescentis by colony hybridization as an indicator of human faecal pollution

AIMS: To develop an improved method for the detection of Bifidobacterium adolescentis as an indicator of human faecal pollution. METHODS AND RESULTS: Bifidobacterium medium (BFM) was identified as the optimal medium for the recovery of bifidobacteria from human effluent. Dilutions of faeces and effluent from both humans and animals were filtered, grown on BFM and human specific B. adolescentis identified via colony hybridization with a digoxigenin (DIG)-labelled oligonucleotide probe. CONCLUSIONS: The combination of BFM with colony probing allows the detection of B. adolescentis, a specific indicator of human faecal pollution. SIGNIFICANCE AND IMPACT OF THE STUDY: It is now technically feasible to use B. adolescentis as indicators of human faecal pollution, and studies to examine the survival and appropriateness of bifidobacteria in this role can be initiated.     

Inhibiting caspase cleavage of huntingtin reduces toxicity and aggregate formation in neuronal and nonneuronal cells

Huntington's disease is a neurodegenerative disorder caused by CAG expansion that results in expansion of a polyglutamine tract at the extreme N terminus of huntingtin (htt). htt with polyglutamine expansion is proapoptotic in different cell types. Here, we show that caspase inhibitors diminish the toxicity of htt. Additionally, we define htt itself as an important caspase substrate by generating a site-directed htt mutant that is resistant to caspase-3 cleavage at positions 513 and 530 and to caspase-6 cleavage at position 586. In contrast to cleavable htt, caspase-resistant htt with an expanded polyglutamine tract has reduced toxicity in apoptotically stressed neuronal and nonneuronal cells and forms aggregates at a much reduced frequency. These results suggest that inhibiting caspase cleavage of htt may therefore be of potential therapeutic benefit in Huntington's disease.     

Mannose-binding lectin-deficient mice display defective apoptotic cell clearance but no autoimmune phenotype

Mannose-binding lectin (MBL) is a circulating serum protein that is sequestered to sites of inflammation and infection. MBL is a member of the collectin family with structural similarities to the lung collectins and functional similarities to C1q. Both MBL and C1q activate complement; C1q activates the classical pathway and MBL the lectin pathway. Here we demonstrate that MBL binds apoptotic cells in vitro and confirm a role for MBL in clearance of apoptotic cells in vivo. Despite MBL null mice demonstrating defective apoptotic cell clearance they did not develop spontaneous autoimmunity, lymphoproliferation, or germinal center expansion although increased numbers of peritoneal B1 cells were detected. These data demonstrate an important in vivo role for MBL in clearance of dying cells and adds the MBL null animals to the few animals with demonstrable in vivo apoptotic cell clearance defects. Moreover, it demonstrates that failure of apoptotic cell clearance can be dissociated from autoimmunity.     

Nonphlogistic clearance of late apoptotic neutrophils by macrophages: efficient phagocytosis independent of beta 2 integrins

Neutrophils undergo constitutive death by apoptosis, leading to safe nonphlogistic phagocytosis and clearance by macrophages. Recent work has shown that before secondary necrosis, neutrophils exhibiting classical features of apoptosis can progress to a morphologically defined late apoptotic state. However, whether such neutrophils could be safely cleared was unknown. We now report that human late apoptotic neutrophils could be purified from cultured neutrophil populations undergoing constitutive death and were subsequently ingested by human monocyte-derived macrophages by serum-independent mechanisms that did not trigger the release of IL-8 or TNF-alpha. Such ingestion was specifically inhibited by Abs to thrombospondin-1 and the alpha(v)beta(3) vitronectin receptor. Murine bone marrow-derived macrophage phagocytosis of late and early apoptotic neutrophils occurred by similar mechanisms, proceeding with the same efficiency as that observed for wild-type controls when macrophages from [alpha(m)](-/-) or [beta(2)](-/-) mice were used. We conclude that specific nonphlogistic, beta(2) integrin-independent mechanisms involving thrombospondin-1 and alpha(v)beta(3) allow macrophages to ingest late apoptotic neutrophils without eliciting inflammatory cytokine secretion.     

Rapid detection of Listeria monocytogenes in ham samples using immunomagnetic separation followed by polymerase chain reaction

AIMS: To develop a 24-h system for the detection of Listeria monocytogenes in ham. METHODS AND RESULTS: An immunomagnetic separation (IMS) of bacteria directly from ham followed by extraction of DNA and detection using a new multiplex polymerase chain reaction (PCR) was used. The PCR method used one primer pair targeted at the listeriolysin O gene of L. monocytogenes and the other pair for a region of the 23S rRNA genes of Listeria, giving products of 706 and 239 bp, respectively. The combined IMS/PCR was calculated to be capable of detecting as few as 1.1 L. monocytogenes cells g-1 in a 25-g ham sample. CONCLUSION: The process produced acceptable results, but the IMS step is the main barrier to further improvement of sensitivity. The DNA isolation was the most time-consuming step in the process. SIGNIFICANCE AND IMPACT OF THE STUDY: A 24-h test for the presence of L. monocytogenes will be useful to the food industry and significantly assist in the timely investigation of outbreaks.