Activated macrophages direct apoptosis and suppress mitosis of mesangial cells

During inflammation in the glomerulus, the complement of resident myofibroblast-like mesangial cells is regulated by mitosis and apoptosis, but the cellular mechanisms controlling the size of mesangial cell populations have remained obscure. Prompted by studies of development, we sought evidence that macrophages regulate mesangial cell number. Rat bone marrow-derived macrophages primed with IFN-gamma then further activated in coculture with LPS or TNF-alpha elicited a 10-fold induction of rat mesangial cell apoptosis and complete suppression of mitosis, effects inhibitable by the NO synthase inhibitors L-monomethyl arginine and L-N(6)-(1-iminoethyl) lysine dihydrochloride. Complete dependence upon macrophage-derived NO was observed in comparable experiments employing activated bone marrow macrophages from wild-type and NO synthase 2(-/-) mice. Nevertheless, when mesangial cells were primed with IFN-gamma plus TNF-alpha, increased induction by activated macrophages of mesangial apoptosis exhibited a NO-independent element. The use of gld/gld macrophages excluded a role for Fas ligand in this residual kill, despite increased expression of Fas and increased susceptibility to soluble Fas ligand exhibited by cytokine-primed mesangial cells. Finally, activated macrophages isolated from the glomeruli of rats with nephrotoxic nephritis also induced apoptosis and suppressed mitosis in mesangial cells by an L-monomethyl arginine-inhibitable mechanism. These data demonstrate that activated macrophages, via the release of NO and other mediators, regulate mesangial cell populations in vitro and may therefore control the mesangial cell complement at inflamed sites.     

Enumeration of Campylobacter in New Zealand recreational and drinking waters

AIMS: To use a published polymerase chain reaction (PCR) method for the detection and identification of thermotolerant Campylobacter species (Camp. jejuni, Camp. coli and Camp. lari) in tandem with a Most Probable Number (MPN) technique to enumerate these species in water samples. METHODS AND RESULTS: An initial study of 42 river water samples compared the use of conventional culture and PCR methods for the detection of Campylobacter in MPN enrichment tubes. It was found that all samples positive by culture were also positive by PCR. Thirty-seven percent more MPN tubes were positive by PCR compared with culture. The MPN/PCR technique was subsequently applied to 96 additional samples collected from rivers, drinking, roof and shallow ground water. Campylobacter was especially prevalent in river water (60% positive) and shallow ground water (75% positive) samples. Drinking water (29.2% positive) and roof water (37.5% positive) also contained Campylobacter, but the numbers detected were very low (maximum 0.3 and 0.56 MPN 100 ml-1, respectively). CONCLUSION: River waters contained Campylobacter at higher levels than any other water type and in a high percentage of the samples. Although Campylobacter was present in treated drinking water, the levels detected were low. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that water may act as a significant transmission route for human campylobacteriosis.     

Causes of variation in malaria infection dynamics: insights from theory and data

Parasite strategies for exploiting host resources are key determinants of disease severity (i.e., virulence) and infectiousness (i.e., transmission between hosts). By iterating the development of theory and empirical tests, we investigated whether variation in parasite traits across two genetically distinct clones of the rodent malaria parasite, Plasmodium chabaudi, explains differences in within-host infection dynamics and virulence. First, we experimentally tested key predictions of our earlier modeling work. As predicted, the more virulent genotype produced more progeny parasites per infected cell (burst size), but in contrast to predictions, invasion rates of red blood cells (RBCs) did not differ between the genotypes studied. Second, we further developed theory by confronting our earlier model with these new data, testing a new set of models that incorporate more biological realism, and developing novel theoretical tools for identifying differences between parasite genotypes. Overall, we found robust evidence that differences in burst sizes contribute to variation in dynamics and that differential interactions between parasites and host immune responses also play a role. In contrast to previous work, our model predicts that RBC age structure is not important for explaining dynamics. Integrating theory and empirical tests is a potentially powerful way of progressing understanding of disease biology.     

Explaining observed infection and antibody age-profiles in populations with urogenital schistosomiasis

Urogenital schistosomiasis is a tropical disease infecting more than 100 million people in sub-Saharan Africa. Individuals in endemic areas endure repeated infections with long-lived schistosome worms, and also encounter larval and egg stages of the life cycle. Protective immunity against infection develops slowly with age. Distinctive age-related patterns of infection and specific antibody responses are seen in endemic areas, including an infection 'peak shift' and a switch in the antibody types produced. Deterministic models describing changing levels of infection and antibody with age in homogeneously exposed populations were developed to identify the key mechanisms underlying the antibody switch, and to test two theories for the slow development of protective immunity: that (i) exposure to dying (long-lived) worms, or (ii) experience of a threshold level of antigen, is necessary to stimulate protective antibody. Different model structures were explored, including alternative stages of the life cycle as the main antigenic source and the principal target of protective antibody, different worm survival distributions, antigen thresholds and immune cross-regulation. Models were identified which could reproduce patterns of infection and antibody consistent with field data. Models with dying worms as the main source of protective antigen could reproduce all of these patterns, but so could some models with other continually-encountered life stages acting as the principal antigen source. An antigen threshold enhanced the ability of the model to replicate these patterns, but was not essential for it to do so. Models including either non-exponential worm survival or cross-regulation were more likely to be able to reproduce field patterns, but neither of these was absolutely required. The combination of life cycle stage stimulating, and targeted by, antibody was found to be critical in determining whether models could successfully reproduce patterns in the data, and a number of combinations were excluded as being inconsistent with field data.     

The trigger to cell death determines the efficiency with which dying cells are cleared by neighbours.

Phagocytosis of apoptotic cells is required to prevent tissue injury. Professional phagocytes, such as monocyte-derived macrophages, are highly efficient scavengers of apoptotic cells but their presence cannot always be relied on; in that case, removal of effete cells is accomplished by helpful neighbours. This study describes differences in the efficiency with which apoptotic cells of the same type, but dying in response to different triggers, are engulfed; this varies from engulfment that is so proficient few or no unengulfed apoptotic cells are found, to engulfment that is so delayed apoptotic cells have become secondarily necrotic at the point of engulfment. In all cases the efficiency of engulfment is determined at least in part by the dying cells themselves. p53- and Bax-transfected kidney epithelial (293) cells (transiently transfected using a non-toxic method) were engulfed so proficiently by homotypic neighbours that cells did not show evidence of engagement of the apoptotic programme (chromatin condensation and TUNEL positivity) until engulfment had taken place. Engulfment nonetheless required activation of at least initiator caspases. 293 cells induced to apoptose by other means (etoposide and staurosporine treatment) were not so efficiently ingested: unengulfed apoptotic cells were consistently revealed at all doses and time points, even when treated cells were mixed with healthy, non-treated 293 cells. These data make it extremely unlikely that the fraction of viable, unaffected neighbours determines the efficiency with which engulfment proceeds. Furthermore, 293 cells treated with etoposide or staurosporine were differentially appealing both to homotypic neighbours and to cells in the professional phagocyte lineage (THP-1 cells). If different apoptotic stimuli programme cells to be recognised with different efficiencies, pathways to apoptosis may be injury limiting to greater or lesser degrees.     

Apoptosis in human eosinophils. Programmed cell death in the eosinophil leads to phagocytosis by macrophages and is modulated by IL-5.

Eosinophils are believed to injure tissues in a variety of allergic disease by virtue of their highly histotoxic contents and metabolites. They are readily observed in tissues during the allergic response yet the mechanisms governing the duration of tissue residence and route of removal remain obscure. We have previously reported in vitro and in vivo evidence that neutrophils undergo apoptosis (programmed cell death) and are recognized and ingested as intact cells by macrophages. We report that eosinophils, purified from the peripheral blood of asymptomatic healthy atopics, undergo apoptosis in vitro. After 72 to 96 h in culture, 57.0 +/- 6.2% (mean +/- SE) of the eosinophil population showed characteristic morphologic changes of apoptosis. Electrophoresis of the DNA from these cells demonstrated the typical "ladder" pattern of internucleosomal DNA cleavage, the hallmark of apoptosis-associated endonuclease activation. The rate of eosinophil apoptosis, slower than that reported for neutrophils, was delayed (by 80 +/- 6 h) in the presence of recombinant human IL-5, a cytokine previously reported to prolong eosinophil life in vitro but not known to modulate apoptosis. Aged, apoptotic eosinophils, but not fresh or aged preapoptotic eosinophils, were recognized and ingested as intact cells by macrophages. Apoptosis and ingestion by macrophages may represent a mechanism whereby the tissue longevity and removal of eosinophils is controlled.     

Glucocorticoids promote nonphlogistic phagocytosis of apoptotic leukocytes

Phagocyte recognition, uptake, and nonphlogistic degradation of neutrophils and other leukocytes undergoing apoptosis promote the resolution of inflammation. This study assessed the effects of anti-inflammatory glucocorticoids on this leukocyte clearance mechanism. Pretreatment of "semimature" 5-day human monocyte-derived macrophages (M phi) for 24 h with methylprednisolone, dexamethasone, and hydrocortisone, but not the nonglucocorticoid steroids aldosterone, estradiol, and progesterone, potentiated phagocytosis of apoptotic neutrophils. These effects were specific in that the potentiated phagocytosis of apoptotic neutrophils was completely blocked by the glucocorticoid receptor antagonist RU38486, and glucocorticoids did not promote 5-day M phi ingestion of opsonized erythrocytes. Similar glucocorticoid-mediated potentiation was observed with 5-day M phi uptake of alternative apoptotic "targets" (eosinophils and Jurkat T cells) and in uptake of apoptotic neutrophils by alternative phagocytes (human glomerular mesangial cells and murine M phi elicited into the peritoneum or derived from bone marrow). Importantly, methylprednisolone-mediated enhancement of the uptake of apoptotic neutrophils did not trigger the release of the chemokines IL-8 and monocyte chemoattractant protein-1. Furthermore, longer-term potentiation by methylprednisolone was observed in maturing human monocyte-derived M phi, with greater increases in 5-day M phi uptake of apoptotic cells being observed the earlier glucocorticoids were added during monocyte maturation into M phi. We conclude that potentiation of nonphlogistic clearance of apoptotic leukocytes by phagocytes is a hitherto unrecognized property of glucocorticoids that has potential implications for therapies aimed at promoting the resolution of inflammatory diseases.     

Competition and dispersal in predator-prey waves

Dispersing predators and prey can exhibit complex spatio-temporal wave-like patterns if the interactions between them cause oscillatory dynamics. We study the effect of these predator-prey density waves on the competition between prey populations and between predator populations with different dispersal strategies. We first describe 1- and 2-dimensional simulations of both discrete and continuous predator-prey models. The results suggest that any population that diffuses faster, disperses farther, or is more likely to disperse will exclude slower diffusing, shorter dispersing, or less likely dispersing populations, everything else being equal. It also appears that it does not matter whether time, space, or state are discrete or continuous, nor what the exact interactions between the predators and prey are. So long as waves exist the competition between populations occurs in a similar fashion. We derive a theory that qualitatively explains the observed behaviour and calculate approximate analytical solutions that describe, to a reasonable extent, these behaviours. Predictions about the cost of dispersal are tested. If strong enough, cost can reverse the populations' relative competitive strengths or lead to coexistence because of the effect of spiral wave cores. The theory is also able to explain previous results of simulations of coexistence in host-parasitoid models (Comins, H. N., and Massell, M. P., 1996, J. Theor. Biol. 183, 19-28).     

Application of polymerase chain reaction (PCR) and TaqMan PCR techniques to the detection and identification of Rhodococcus coprophilus in faecal samples.

Rhodococcus coprophilus, a natural inhabitant of herbivore faeces, has been suggested as a good indicator of animal (as opposed to human) faecal contamination of aquatic environments. However, conventional detection methods limit its use for this as they require up to 21 days to obtain a result. In this paper an optimised method for extracting R. coprophilus DNA from faecal samples is described. PCR and 5'-nuclease (TaqMan) PCR methods were developed to allow the detection and enumeration of R. coprophilus in faecal samples within 2-3 days. Both PCR methods targeted the 16S rRNA gene, producing an amplicon of 443 bp which was specific for R. coprophilus. Sixty cells were required to produce an amplification product by conventional PCR, while as little as one cell was required for the TaqMan PCR method. The latter approach gave a linear quantitative response over at least four log units with both bacterial cells and DNA. Successful amplification by PCR was achieved using DNA extracted from cow, sheep, horse and deer faeces but was negative for samples from humans, pig, possum, duck and rabbit. These PCR methods enhance the feasibility of using R. coprophilus to distinguish faecal pollution of farmed herbivores from human pollution.     

Conditional macrophage ablation demonstrates that resident macrophages initiate acute peritoneal inflammation

The role played by resident macrophages (Mphi) in the initiation of peritoneal inflammation is currently unclear. We have used a conditional Mphi ablation strategy to determine the role of resident peritoneal Mphi in the regulation of neutrophil (PMN) recruitment in experimental peritonitis. We developed a novel conditional Mphi ablation transgenic mouse (designated CD11bDTR) based upon CD11b promoter-mediated expression of the human diphtheria toxin (DT) receptor. The murine DT receptor binds DT poorly such that expression of the human receptor confers toxin sensitivity. Intraperitoneal injection of minute (nanogram) doses of DT results in rapid and marked ablation of F4/80-positive Mphi populations in the peritoneum as well as the kidney, and ovary. In experimental peritonitis, resident Mphi ablation resulted in a dramatic attenuation of PMN infiltration that was rescued by the adoptive transfer of resident nontransgenic Mphi. Attenuation of PMN infiltration was associated with diminished CXC chemokine production at 1 h. These studies indicate a key role for resident peritoneal Mphi in sensing perturbation to the peritoneal microenvironment and regulating PMN infiltration.