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In deserts, the interplay between occasional fluvial events and persistent aeolian erosion can form composite modern and relict surfaces, especially on the distal portion of alluvial fans. There, relief inversion of alluvial deposits by differential erosion can form longitudinal ridges. We identified two distinct ridge types formed by relief inversion on converging alluvial fans in the hyperarid Chilean Atacama Desert. Although they are co-located and similar in scale, the ridge types have different ages and formation histories that apparently correspond to minor paleoclimate variations. Gravel-armored ridges are remnants of deflated alluvial deposits with a bimodal sediment distribution (gravel and sand) dated to a minor pluvial phase at the end of the Late Pleistocene (~12 kyr). In contrast, younger (~9 kyr) sulfate-capped ridges formed during a minor arid phase with evaporite deposition in a pre-existing channel that armored the underlying deposits. Collectively, inverted channels at Salar de Llamara resulted from multiple episodes of surface overland flow and standing water spanning several thousand years. Based on ridge relief and age, the minimum long-term deflation rate is 0.1-0.2 m/kyr, driven primarily by wind erosion. This case study is an example of the equifinality concept whereby different processes lead to similar landforms. The complex history of the two ridge types can only be generally constrained in remotely sensed data. In situ observations are required to discern the specifics of the aqueous history, including the flow type, magnitude, sequence, and paleoenvironment. These findings have relevance for interpreting similar landforms on Mars.

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The U.S. National Institute for Occupational Safety and Health (NIOSH) Office of Mine Safety and Health Research (OMSHR) conducted a study of mining industry electrical injuries reported to the U.S. Mine Safety and Health Administration (MSHA) for the years 2000 to 2009. The findings of that study are detailed in this paper, and serve to characterize the circumstances surrounding electrical injuries and identify causal factors. The study included three tasks: 1) a direct review of mining industry occupational injury data compiled by MSHA, 2) interpretation of the narrative descriptions available for the injuries (from MSHA data) and 3) a separate examination of fatal electrical injuries. Eight-hundred sixty-five electrical injuries were reported during the 10-year period studied, with 39 of those being fatalities. This makes electrical injuries disproportionately fatal with respect to most other types of injuries in mining. Electrical injury rates were higher in coal mining than noncoal mining and, within the coal sector, rates were higher in underground operations than in surface operations. Of the 865 total cases, electrical and machine maintenance or repair activities were involved in 580 (69%), and electricians and mechanics were injured in 362 cases (42%). Of the 39 fatal electrical injuries, 27 (69%) involved electrical maintenance or repair work, and in 21 of these 27 cases, the failure to de-energize, lock-out and tag the circuit was the cause or a contributing factor. Also, contractor employees had a much greater chance of an electrical injury being fatal than did mine operator employees. The top three root causes for fatal electrical injuries were 1) no or inadequate lock-out and tagging, 2) failure of power system components and 3) contact of overhead electrical power lines by mobile equipment.

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HCs are clonal late B cells that are related to memory cells and display specific features of activation. Many of the distinctive features of HCs (eg, morphology, TRAP) are related to this specific activation. Many of the distinctive histologic features of HCL can be related to constitutive production of cytokines (eg, FGF, fibrosis) and to the expression/activation of adhesion receptors (eg, alpha(4)beta(1), alpha(5)beta(1) and alpha(v)beta(3) integrins, CD44v3). HCs usually have mutated IGVH genes and have no consistent or specific chromosome abnormalities (5q additions and 7q deletions in a minority). The signals that are responsible for several of the phenotypic features of HCs have been identified, but the nature of the underlying oncogenic events remains unknown.

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CD154 is an important regulator of chronic lymphocytic leukaemia (CLL)-cell survival. In CLL, high serum levels of VEGF are a feature of advanced disease, and we and others have previously shown that CLL cells produce and secrete this growth factor. Since CD154 stimulates VEGF production in other cell types, and VEGF is known to promote cell survival, we examined whether the cytoprotection of CLL cells by CD154 involves VEGF. We report for the first time that treatment of CLL cells with CD154 results in increased VEGF production and demonstrate involvement of NF-kappaB in this process. Moreover, we show that CD154-induced CLL-cell survival is reduced by anti-VEGF-neutralising antibody and by inhibiting VEGF receptor (VEGFR) signalling with SU5416. However, addition of exogenous VEGF alone or blocking secreted autocrine VEGF had little or no effect on CLL-cell survival. We therefore conclude that CLL-cell cytoprotection in the presence of CD154 requires combined signalling by both CD40 and VEGFR. This combined signalling and resulting cytoprotection were shown to involve NF-kappaB activation and increased survivin production. In conclusion, our findings identify autocrine VEGF as an important mediator of the antiapoptotic effect of CD40 ligation, and thus provide new insights into CLL-cell rescue by CD154 in lymphoreticular tissues.

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Hairy-cell leukaemia (HCL) is usually readily diagnosed by seeing typical hairy cells (HCs) in the blood film. The diagnosis is then confirmed by tartrate-resistant acid phosphatase staining, marker analysis, and bone marrow examination. HCs are clonal mature memory B cells with specific features of activation. This HC activation is responsible for many of the pathological features of the disease, including its distinctive bone marrow fibrosis, splenic red pulp invasion, and pseudo-sinus formation. Chlorodeoxyadenasine is the treatment of first choice. Deoxycoformycin and rituximab are useful for the treatment of relapsed/refractory disease. The nature of the primary oncogenic event(s) remains unknown and is the major unresolved issue in HCL.

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The evidence that hairy cells are activated clonal late B cells is presented. The largely non-specific (i.e. not confined to hairy-cell leukaemia) chromosome and genetic abnormalities are then described. Next, the features of malignant-cell activation are considered, including the distinctive morphology of hairy cells and their expression of activation-related antigens and activated adhesion receptors. Also, signalling and cytokine production are discussed in the context of malignant-cell activation. It is then demonstrated that many of the distinctive clinicopathological features of hairy-cell leukaemia can be explained in terms of the interaction of the activated malignant cells with other types of cell and tissue matrix. Finally, the biological basis of the hairy cell's unusually high sensitivity to IFN-alpha and nucleoside analogues is discussed.

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The diagnosis of HCL is usually straightforward and is based on the identification of typical HCs in the blood and bone marrow. The suspected diagnosis is confirmed by a combination of TRAP cytochemistry, a distinctive immunophenotype and characteristic BM trephine appearances. Nucleoside treatment is highly effective in inducing prolonged remissions; relapsing patients can usually be successfully retreated with nucleoside. Monoclonal antibody therapy is a promising novel approach to the treatment of resistant disease.

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Chronic lymphocytic leukaemia (CLL) cells are long-lived in vivo but undergo spontaneous apoptosis when cultured in vitro. Since CLL cells associate intimately with one another at sites of tissue involvement, we examined the hitherto unproven possibility that homotypic interactions between the malignant cells might reduce their propensity to undergo spontaneous cell death. In a series of experiments in which highly pure CLL-cell populations were cultured on a non-adherent surface, cell viability was found to increase markedly with the level of crowding at the bottom of the culture vessel. The effect was observed among unevenly distributed cells within a single culture vessel and did not require direct cell-cell contact. This indicates that cell survival was being regulated in an autocrine fashion by locally acting soluble products. Conditioned medium from crowded CLL cells enhanced the survival of autologous non-crowded cells, indicating that at least some of the autocrine survival factors produced by CLL cells could accumulate in the extracellular environment. In addition, the survival of non-crowded CLL cells was markedly enhanced by co-culturing them with an excess of autologous fixed cells. This protective effect of direct cell-cell contact was mediated by specific surface structures since it was abrogated by pre-treating the fixed cells with neuraminidase. Our results provide the first direct demonstration that the survival of cultured CLL cells is enhanced by homotypic interactions. We speculate that these protective effects may contribute to the accumulation of CLL cells in vivo, and that further elucidation of the underlying mechanisms may lead to novel therapeutic strategies.

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The well-established association between TP53 mutations and adverse clinical outcome in a range of human cancers reflects the importance of p53 protein in regulating tumor-cell growth and survival. Although it is theoretically possible for p53 dysfunction to arise through mechanisms that do not involve TP53 mutation, such a phenomenon has not previously been demonstrated in a sporadic tumor. Here, we show that p53 dysfunction in B-cell chronic lymphocytic leukemia (CLL) can occur in the absence of TP53 mutation and that such dysfunction is associated with mutation of the gene encoding ATM, a kinase implicated in p53 activation. Forty-three patients with CLL were examined for p53 dysfunction, as detected by impaired up-regulation of p53 and of the p53-dependent protein p21(CIP1/WAF1) after exposure to ionizing radiation (IR). Thirty (70%) patients had normal p53 responses and underwent progressive IR-induced apoptosis. In 13 (30%) patients, p21 up-regulation was markedly impaired, indicating p53 dysfunction. Six (14%) of these patients with p53 dysfunction had increased baseline levels of p53, were found to have TP53 mutations, and were completely resistant to IR-induced apoptosis. In the other 7 (16%) patients with p53 dysfunction, IR-induced p53 up-regulation and apoptosis were markedly impaired, but baseline levels of p53 were not increased, and no TP53 mutations were detected. Each of these patients was found to have at least one ATM mutation, and a variable reduction in ATM protein was detected in all 4 patients examined. This is the first study to provide a direct demonstration that p53 dysfunction can arise in a sporadic tumor by a mechanism that does not involve TP53 mutation. (Blood. 2001;98:814-822)

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The tissue homing of malignant hematic cells has both diagnostic and pathogenetic importance. Although such homing is incompletely understood, it generally involves cell adhesion and migration mediated by a number of adhesion receptors and cytokines. In this article, the potential importance of hyaluronan (HA) is examined for the tissue homing of hairy cells (HCs) in hairy cell leukemia (HCL). It is shown that HCs readily adhere to, and spontaneously move on, HA-coated surfaces using CD44. This indicates that activated CD44 and spontaneous movement on HA form part of the intrinsically activated phenotype of HCs. Interleukin-8 (IL-8) inhibited HC movement on HA, and this cell arrest was accompanied by increased actin polymerization and a more pronounced association of CD44 with the cytoskeleton. All of these findings are in sharp contrast to our previous observations with chronic lymphocytic leukemia cells, which are nonmotile on HA, but in response to IL-8 become polarized and motile using the receptor for HA-mediated motility rather than their apparently inactive CD44. Immunohistochemical examination of HCL tissues showed the ubiquitous presence of IL-8 and the prominence of HA in bone marrow stroma and hepatic portal tracts. This suggests that CD44-HA interactions are important in HC homing to these sites, but not to splenic red pulp or hepatic sinusoids, where HA is largely absent. Moreover, engagement of CD44 on HCs stimulates fibronectin synthesis, an observation that is likely to be relevant to the restriction of fibrosis in the disease to HC-infiltrated areas containing HA.

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Expansion of primary solid tumors and their malignant dissemination are angiogenesis-dependent. Vascular endothelial growth factor (VEGF) is the key factor playing a pivotal role in solid tumor-induced angiogenesis. Recent studies indicate that angiogenesis may also be involved in the pathogenesis of certain hemic malignancies, including B-cell chronic lymphocytic leukemia (B-CLL). Mechanisms underlying angiogenesis in B-CLL and the role of VEGF in this process are incompletely understood. In this study, it was examined whether angiogenically functional VEGF is produced by B-CLL cells. Immunohistochemical staining with antibodies against VEGF and CD34, an endothelial cell marker, demonstrated the presence of VEGF protein and abundant blood vessels in infiltrated lymphoreticular tissues. Low levels of VEGF were detected by ELISA in the culture media of unstimulated cells; this was enhanced up to 7-fold by hypoxic stimulation. SDS-PAGE and Western blot analysis of the concentrated culture media showed 2 isoforms of VEGF protein with molecular weights of 28 and 42 kd, respectively. RNA hybridization showed that these cells expressed VEGF mRNA. Reverse transcription-polymerase chain reaction, combined with nucleotide sequence analysis, revealed that the predominantly expressed isoforms were VEGF121 and VEGF165. Moreover, (3)H-thymidine incorporation and an in vivo angiogenic assay demonstrated that the VEGF produced by CLL cells can induce angiogenesis by stimulating endothelial cell proliferation. In conclusion, this study shows that B-CLL cells produce VEGF and demonstrates the angiogenic effects of this growth factor, which may be relevant for the tissue phase of the disease.

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Caspases are known to be involved in the apoptotic killing of chronic lymphocytic leukaemia (CLL) cells by fludarabine. However, it is unclear whether these enzymes are required for the induction of such killing, or whether they simply determine the mode of cell death. To address this question, we examined the effect of the broad-spectrum caspase inhibitor Z-VAD.fmk on six different manifestations of nucleoside cytotoxicity. Our results indicate that while caspase activity is required for nucleoside-induced poly(ADP-ribose) polymerase (PARP) cleavage and DNA fragmentation, other manifestations of cell death (mitochondrial depolarization, exposure of phosphatidyl serine, cell membrane disruption and cell shrinkage) are caspase independent. By showing that caspases influence the mode, but not the extent, of nucleoside cytotoxicity, our results exclude defects in these enzymes as a mechanism of nucleoside resistance in CLL.

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Although the nucleoside analogues fludarabine and chlorodeoxyadenosine have become important therapeutic agents in chronic lymphocytic leukemia (CLL), their effectiveness is limited by drug resistance. Because such resistance is likely to result from impaired drug-induced apoptosis, it is clearly important to understand the mechanisms involved in this process. Whereas p53 can contribute to the nucleoside-induced killing of CLL cells, recent work from this laboratory and elsewhere has shown that such killing can also occur by p53-independent mechanisms. Because poly(ADP-ribose) polymerase (PARP)-mediated NAD+/ATP depletion has been implicated in the nucleoside-induced killing of normal resting lymphocytes, we postulated that this mechanism might account for the p53-independent component of nucleoside cytotoxicity in CLL. To address this question, we used 3-aminobenzamide (3AB) at a concentration (200 microM) known to produce selective inhibition of poly(ADP-ribosyl)ation in intact cells and examined nucleoside-induced killing using a number of different end points (cell membrane disruption, cell shrinkage, mitochondrial depolarization, exposure of phosphatidyl serine, morphological changes, DNA fragmentation, and PARP-1 cleavage). In 27 of the 30 cases of CLL examined, 3AB delayed nucleoside-induced cell membrane disruption without inhibiting other manifestations of cytotoxicity. This indicates that PARP activity, rather than contributing to the induction of cell killing, was accelerating cell membrane disruption during the late stages of apoptosis. This novel observation has important implications for previous studies of PARP-mediated cytotoxicity. However, in cells from one CLL patient, 3AB inhibited all manifestations of nucleoside cytotoxicity; this was the only case in the study known to have a p53 gene defect affecting both alleles. This indicates that PARP activity can occasionally be central to nucleoside-induced killing and that such PARP-mediated killing is p53 independent.

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The role of GM-CSF in B cell (patho)physiology is unclear. Although B cells can respond to GM-CSF, there is controversy concerning the extent to which various resting and activated B cell types can themselves produce this cytokine, and the possibility that it can function in an autocrine fashion has not previously been considered. The aim of the present study was to address these issues using hairy cells (HCs) and chronic lymphocytic leukemia cells, two intrinsically activated mature malignant B cell types (with activation being more uniform and more pronounced in HCs). Normal B cells were used for comparison. Using a number of techniques, we demonstrated the constitutive production of GM-CSF by all three cell types and showed that the cytokine was biologically active. GM-CSF mRNA and protein were increased after cell activation by PMA, and constitutive production of the cytokine was highest in HCs, suggesting that the level of GM-CSF production is influenced by cell activation. Because GM-CSF is known to be antiapoptotic for myeloid cells, we used blocking anti-GM-CSF Abs to examine the contribution of autocrinely produced cytokine to cell survival. The Abs produced marked reduction in the in vitro survival of HCs, chronic lymphocytic leukemia cells, and normal B cells by promoting apoptosis. Taken together, these findings suggest that, in combination with other known rescue factors, autocrinely produced GM-CSF may contribute to normal and malignant B cell survival in vivo.

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To resolve the controversy concerning the role of p53 in the killing of resting lymphocytes by purine nucleoside analogues, we examined the cytotoxic effects of chlorodeoxyadenosine, fludarabine and deoxycoformycin (plus deoxyadenosine) on unstimulated spleen cells from p53-knockout versus wild-type mice. p53-knockout cells were more resistant to all three nucleosides than were wild-type cells. However, substantial killing still occurred in the absence of p53, indicating that purine analogues can kill resting lymphocytes by both p53-dependent and -independent mechanisms. We suggest that these results are relevant to chronic lymphoid malignancies, and that characterization of the p53-independent component of nucleoside action may indicate potential ways of overcoming therapeutic resistance.

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To clarify the role of p53 in the killing of chronic lymphocytic leukaemia (CLL) cells by purine analogues, we examined the cytotoxic effects of chlorodeoxyadenosine and fludarabine on CLL cells that had been characterized according to their p53 functional status. Cases of CLL with p53 dysfunction (n = 7) displayed slight, but significant, resistance to nucleoside-induced cell killing when compared with cases with functionally intact p53 (n = 12). The small difference between the two groups indicated that p53 plays a minor role in such killing. These findings suggest that the poor therapeutic response to purine analogues observed in patients with p53 defects is likely to be caused by the emergence, on a background of genomic instability, of CLL-cell clones that are resistant to nucleoside-induced killing for reasons unrelated to p53.

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Malignant lymphocyte migration into and within lymphoreticular tissue is an important aspect of chronic lymphocytic leukemia (CLL), yet little is known about the processes involved. Our previous studies of integrin expression and function in CLL have shown that the abnormal cells are relatively nonadhesive and nonmotile on the protein ligands of these receptors. Here we show that CLL cells adhere to a non-protein ligand, hyaluronan (HA), and become motile (as assessed by both Boyden chamber migration and time-lapse video microscopy) on this ligand when stimulated with interleukin (IL) 8. The combined presence of HA and IL-8 was essential for this motility because IL-8 did not stimulate movement on other surfaces. Blocking antibodies showed that this motility is mediated by the receptor for HA-mediated motility (RHAMM), without the involvement of CD44. Moreover, confocal microscopy showed a polarized distribution of RHAMM and F-actin, but not CD44, in cells which had become motile on HA in the presence of IL-8. Immunohistochemical studies of nodes and spleen demonstrated an abundant reticular network of HA-containing fibers throughout diseased nodes and in splenic white pulp. The splenic red pulp and the luminal surface of high endothelial venules lacked HA. IL-8 was ubiquitously present in these tissues. CLL cells were shown to move spontaneously on fibroblast monolayers derived from lymphoid tissue; this movement was largely blocked by hyaluronidase or anti-RHAMM or anti-IL-8 antibodies. These studies indicate that IL-8-induced motility on HA is likely to be important for CLL cell migration through lymphoid tissue.

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