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[Leishmania(L.)] amazonensis amastigotes reside in macrophages within spacious parasitophorous vacuoles (PVs) which may contain numerous parasites. After sporadic fusion events were detected by time-lapse cinemicrography, PV fusion was examined in two different models. In single infections, it was inferred from the reduction in PV numbers per cell. In a reinfection model, macrophages infected with unlabeled amastigotes were reinfected with GFP-transfected- or carboxyfluorescein diacetate succinimidyl ester-labeled parasites, and fusion was detected by the colocalization of labeled and unlabeled amastigotes in the same PVs. The main findings were: (1) as expected, fusion frequency increased with the multiplicity of infection; (2) most fusion events took place in the first 24h of infection or reinfection, prior to the multiplication of incoming parasites; (3) resident and incoming parasites multiplied at similar rates in fused PVs. The model should be useful in studies of parasite and host cell factors and mechanisms involved in PV fusogenicity.

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Invasive bacteria can induce their own uptake and specify their intracellular localization; hence it is commonly assumed that proximate modulation of host cell transcription is not required for infection. However, bacteria can also modulate, directly or indirectly, the transcription of many host cell genes, whose role in the infection may be difficult to determine by global gene expression. Is the host cell nucleus proximately required for intracellular infection and, if so, for which pathogens and at what stages of infection? Enucleated cells were previously infected with Toxoplasma gondii, Chlamydia psittaci, C. trachomatis, or Rickettsia prowazekii. We enucleated L929 mouse fibroblasts by centrifugation in the presence of cytochalasin B, and compared the infection with Shigella flexneri M90T 5a of nucleated and enucleated cells. Percent infection and bacterial loads were estimated with a gentamicin suppression assay in cultures fixed and stained at different times after infection. Enucleation reduced by about half the percent of infected cells, a finding that may reflect the reduced endocytic ability of L929 cytoplasts. However, average numbers of bacteria and frequency distributions of bacterial numbers per cell at different times were similar in enucleated and nucleated cells. Bacteria with actin-rich tails were detected in both cytoplasts and nucleated cells. Lastly, cytoplasts were similarly infected 2 and 24 h after enucleation, suggesting that short-lived mRNAs were not involved in the infection. Productive S. flexneri infection could thus take place in cells unable to modulate gene transcription, RNA processing, or nucleus-dependent signaling cascades.

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Invasive bacteria can induce their own uptake and specify their intracellular localization; hence it is commonly assumed that proximate modulation of host cell transcription is not required for infection. However, bacteria can also modulate, directly or indirectly, the transcription of many host cell genes, whose role in the infection may be difficult to determine by global gene expression. Is the host cell nucleus proximately required for intracellular infection and, if so, for which pathogens and at what stages of infection? Enucleated cells were previously infected with Toxoplasma gondii, Chlamydia psittaci, C. trachomatis, or Rickettsia prowazekii. We enucleated L929 mouse fibroblasts by centrifugation in the presence of cytochalasin B, and compared the infection with Shigella flexneri M90T 5a of nucleated and enucleated cells. Percent infection and bacterial loads were estimated with a gentamicin suppression assay in cultures fixed and stained at different times after infection. Enucleation reduced by about half the percent of infected cells, a finding that may reflect the reduced endocytic ability of L929 cytoplasts. However, average numbers of bacteria and frequency distributions of bacterial numbers per cell at different times were similar in enucleated and nucleated cells. Bacteria with actin-rich tails were detected in both cytoplasts and nucleated cells. Lastly, cytoplasts were similarly infected 2 and 24 h after enucleation, suggesting that short-lived mRNAs were not involved in the infection. Productive S. flexneri infection could thus take place in cells unable to modulate gene transcription, RNA processing, or nucleus-dependent signaling cascades.

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Coxiella burnetii, the agent of Q fever in man and of coxiellosis in other species, is an intracellular pathogen not yet grown axenically. Confocal laser fluorescence microscopy and morphometry were used to measure relative C. burnetii phase II loads and their intracellular distribution in aldehyde fixed and DAPI stained Vero cell monolayers. The fluorescence of single horizontal optical sections provided useful information on relative loads of bacteria in cells and vacuoles. The relative density of the bacteria in the vacuoles was inferred from ratios of fluorescence to vacuolar section areas. Relative bacterial loads, bacterial densities and section areas of large vacuoles increased exponentially between days 2 and 4 of the infection of gamma-irradiated host cells, stabilized between days 4 and 6, and decreased thereafter. Estimated minimum doubling times were higher for the overall complement of the intracellular organisms (about 12 h) than for bacteria that were confined to larger vacuoles (about 10 h).

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Studies on fixed preparations have shown that vacuoles containing zymosan (Z) particles internalized by infected macrophages can selectively fuse with the large parasitophorous vacuoles (PVs) that shelter Leishmania amazonensis. To examine the kinetics of vacuolar fusion in individual cells, particles were followed by time-lapse cinemicrography from their uptake to their entry in a PV. Newly formed Z-containing vacuoles moved centripetally and, if they contacted a PV, the two vacuoles remained closely apposed for variable, often extended, periods of time before they eventually fused. Transmission electron microscopy confirmed that the cytoplasm separating the partner vacuoles could be reduced to a very thin layer. Initiation of fusion was indicated by reduced refractility of the boundary between Z vacuoles and target PVs. Within a few minutes the PV enlarged and encompassed the Z particles, which remained immobile throughout. The interval between phagocytosis and fusion, 50 +/- 7.4 min (N = 17; range, 4 to 108 min), suggests that most but not all Z vacuoles underwent significant maturation by the time of fusion. Some particles were transferred singly, others entered PVs in groups of 2 or more, and additional clustered transfers to the same vacuole were also observed. These observations provide a baseline for studies of the biochemical mechanisms and the pharmacological control of the fusion of Leishmania PVs, and for the comparison of the fusion behavior of the PVs with that of other phagocytically derived vacuoles.

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Living Leishmania amazonensis amastigotes were incubated with radioiodinated N-benzyloxycarbonyl-L-tyrosyl-L-alanyl diazomethane (Z-Tyr-AlaCHN2), an irreversible inhibitor of mammalian cathepsins B and L. Parasite lysates were subjected to electrophoresis in gelatin-containing sodium dodecyl sulfate-acrylamide gels to detect regions of proteolytic activity, and the distribution of the inhibitor was ascertained by autoradiography. Of the three main bands of proteolysis associated with cysteine proteinases, two, with apparent molecular weights of 28 and 31 kDa, were shown to be labeled. The third enzyme activity, detected at the 35-kDa region in substrate gels, was only faintly labeled. The distribution of labeled bands was similar when lysates of untreated parasites were electrophoresed and the gels incubated with the radioiodinated inhibitor. Under reducing conditions, the inhibitor bound to polypeptides of 29, 31, 32, and 34 kDa, of which the first and the last were the most intensely labeled. Polypeptides with the same apparent molecular weights were labeled when amastigote lysates were incubated with the 125I inhibitor. Uptake of radioactivity by the parasites was time and concentration-dependent and more than 80% of the total counts could be precipitated with trichloroacetic acid. Radioactivity associated with the amastigotes was quite stable after they were pulsed with labeled inhibitor and chased for up to 24 hr in inhibitor-free medium. Both total uptake and labeling of cysteine proteinases were markedly reduced in parasites preincubated with Z-Phe-AlaCHN2 prior to exposure to Z-Tyr(125I)-AlaCHN2. However, more radioiodinated inhibitor was taken up by parasites preincubated with cold inhibitor and chased in inhibitor-free medium, suggesting de novo synthesis or processing of inactive enzyme precursors.

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L-amino acid esters such as leucine methyl ester (Leu-OMe) destroy Leishmania mexicana amazonensis amastigotes by a mechanism which may involve hydrolysis of the compounds by parasite enzymes. Moreover, several esters (e.g. Ile-OMe) prevent the killing of parasites by Leu-OMe, perhaps by inhibition of the hydrolytic enzymes. We show here that certain amino acid amides are also leishmanicidal. Killing of Leishmania within macrophages was assessed microscopically, and that of isolated amastigotes was measured by reduction of the tetrazolium MTT. Amino acid amides were generally less active than the methyl esters and several were more toxic to the macrophages, as determined by inspection of Giemsa-stained preparations. Ranks of activity of the amides on isolated amastigotes were Trp greater than Leu greater than Phe greater than Met greater than Tyr. The amides of Ala, Gly, Val, Ile, His and D-Leu were inactive. This pattern of activity is similar to that of amino acid methyl esters. Ile-NH2 and a few other amides protected intracellular as well as isolated parasites from killing by Leu-OMe. Conversely, Ile-OMe reduced the toxicity of Leu-NH2 for isolated amastigotes. None of the esters or amides assayed prevented the destruction of Leishmania by Trp-NH2. The results are compatible with the view that amino acid esters and amides may be recognized by the same or similar parasite enzymes.

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We carried out a prospective study in 66 infants with congenital diaphragmatic hernia within the first 6 hours of life to determine whether outcome is related to the degree of underlying pulmonary hypoplasia, as predicted by preoperative PaCO2, when correlated with an index of ventilation (VI = mean airway pressure X respiratory rate) and confirmed by postmortem analysis of the lung. Those infants with PaCO2 greater than 40 mm Hg before surgery had a 77% mortality; when PaCO2 reduction could be achieved only with VI greater than 1000, the mortality was still greater than 50%. After repair, however, the ability to hyperventilate to PaCO2 less than 40 mm Hg proved to be an important determinant of survival; only one of 31 infants in this group died, whereas only two of 27 infants with PaCO2 greater than 40 mm Hg survived. In 16 infants with PaCO2 greater than 40 mm Hg despite hyperventilation, high-frequency oscillatory ventilation was started. This resulted in a rapid fall in PaCO2, but 14 of the 16 infants had only temporary improvement in oxygenation, and died. In five of the infants who died, alveolar number was assessed by postmortem morphometric analysis; there was a severe reduction to less than 10% of published normal neonatal values. Pulmonary vascular changes of increased muscularization were less remarkable than those observed in infants with persistent pulmonary hypertension. Our findings suggest that the degree of pulmonary hypoplasia (which would not be influenced by surgical repair), rather than the pulmonary vascular abnormality, mainly determines survival. Consideration could therefore be given to an initial nonsurgical approach to congenital diaphragmatic hernia, with the expectation that pulmonary function might improve and pulmonary vascular resistance decrease.

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L-Amino acid esters, such as leucine methyl ester (Leu-OMe) can destroy intracellular as well as isolated amastigotes of Leishmania mexicana amazonensis by a mechanism which may involve ester hydrolysis by parasite enzymes. We show here that several other esters prevented the killing of the amastigotes by Leu-OMe. Destruction of Leishmania within macrophages in culture was assessed microscopically and viability of isolated parasites was monitored by reduction of the tetrazolium MTT. The main features of the protective effect were similar for intracellular and for isolated amastigotes. Thus, (i) effective prevention of parasite killing required that the protective ester be present in the medium prior to and during exposure of infected cells or parasites to Leu-OMe; (ii) the same esters protected intracellular and isolated Leishmania against damage by Leu-OMe. Ranks of protective activity, as determined on isolated amastigotes were: Gly-OBz greater than Tyr-OMe greater than Ile-OMe greater than Met-OMe greater than Val-OMe greater than Ala-OMe greater than Gly-OMe greater than D-Leu-OMe; (iii) several esters were inactive in both systems (Leu-OBz, Trp-OMe and Phe-OMe). Protective activity was associated with leishmanicidal (e.g. Gly-OBz, Tyr-OMe) as well as with non-leishmanicidal (e.g. Ile-OMe, Val-OMe) esters. The results are compatible with the hypothesis that protective esters inhibit the activity of parasite enzyme(s) which hydrolyse Leu-OMe.

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Amino acid esters can destroy intracellular as well as isolated amastigotes of Leishmania mexicana amazonensis. In the present study we examined, using a tetrazolium reduction assay, the toxicity of the esters for amastigotes isolated from mouse lesions. Parasite killing by the "prototype" compound L-leucine methyl ester at 1 mM concentration and at pH 7.3 took place within 15-30 min. Time-lapse cinematographic observations showed that the amastigotes rounded up and became less phase-dense before they rapidly broke down. Ammonium chloride, ethylamine or monensin, known to raise the intracellular pH, reduced the sensitivity of the amastigotes to L-Leu-OMe. This finding suggests that an acidified compartment is involved in the destruction of the parasites. The leishmanicidal activity of a series of L-amino acid esters was also investigated. The ED50 (concentration for half maximal effect) for methyl esters was (in mM): Leu (0.62), Trp (0.96), Met (1.13), Glu (2.0), Phe (2.5), and Tyr (3.8). In contrast, the methyl esters of Ile, Val, Ala, beta Ala, Gly, Ser, His, and Pro were either inactive or weakly active at 15 mM. Benzyl esters were more active than their methyl homologs: the ED50 of the benzyl esters of Leu, Val, Ile, Gly, Ala, beta Ala, and Pro were, respectively, 0.07, 0.20, 0.22, 0.88, 1.5, 2.3, and 6.7 mM. Ranks of leishmanicidal activity may reflect differences in the rates of ester uptake and trapping by the amastigotes, in the specificity of the relevant hydrolytic enzyme(s), in the accumulation and metabolic fate of the released amino acids, or in the toxicity of the amino acid or alcohol released within the amastigotes.

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Amino acid esters can disrupt lysosomes and damage monocytes and certain lymphocyte populations. Lysosomal disruption involves pH trapping of the esters, followed by their hydrolysis by as yet unidentified enzymes. Accumulation of the more polar amino acids is assumed to cause osmotic lysis of the organelles. We have discovered that certain amino acid esters and amides destroy Leishmania mexicana amazonensis amastigotes lodged within macrophages in culture, as well as parasites isolated from mouse lesions. This paper reviews the amino acid specificity of parasite killing, the resistance of amastigotes derived from infection of macrophages with promastigotes, the involvement of an acidified compartment within the parasites, and the protection conferred by other amino acid esters, and by the protease inhibitors antipain and chymostatin, against the destruction of amastigotes by Leucine-methyl ester. Studies with tritiated esters confirm the critical role of ester hydrolysis for leishmanicidal activity and strengthen the view that similar mechanisms underlie disruption of lysosomes and destruction of Leishmania. Characterization of the parasite organelles and of the enzymes involved in the leishmanicidal activity as well as structure-activity studies may permit the design of compounds more selective for the parasites.

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Amino acid esters can disrupt lysosomes and damage monocytes and certain lymphocyte populations. Lysosomal disruption involves pH trapping of the esters, followed by their hydrolyssis by as yet unidentified enzymes. Accumulation of the more polar amino acids is assumed to cause osmotic lysis of the organelles. we have discovered that certain amino acid esters and amides destroy Leishmania mexicana amazonensis amastigores lodged within macrophages in culture, as well as parasites isolated from mouse lesions. This paper reviews the amino acid specificity of parasite killing, the resistance of amastigotes derived from infection of macrophages with promastigotes, the involvement of an acidified compartment within the parasites, and the protection conferred by other amino acid esters, and the protease inhibitors antipain and chymostatin, aginst the destruction of amastigotes by Leucine-methyl ester. Studies with tritiated esters confirm the critical role of ester hydrolysis for leishmanicidal activity and strengthen the view that similar mechanisms underlie disruption of lysosomes and destruction of Leismania. Characterization of the parasite organelles and of the enzymes involved in the leishmanicidal activity as well as structure-activity studies may permit the design of compounds mor selective for the parasites

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Leishmania amastigotes parasitize almost exclusively the mononuclear phagocytes of mammals. The organisms survive and multiply within acidified vacuoles (parasitophorous vacuoles; p.v.) akin to phagolysosomes. Certain amino acid esters are known to accumulate in and disrupt lysosomes. We postulated that, since Leishmania possess lysosome-like organelles, they may be susceptible to the potentially high ester concentrations attained in the p.v. We report here that L-amino acid esters can rapidly destroy intracellular Leishmania at concentrations that do not appear to damage the host cells. L-leu-OMe, which cured greater than or equal to 90% of infected macrophages at 0.8 mM concentrations, was used in most of the experiments. L-leu-OMe was only active after infection, implying inefficient transfer from secondary lysosomes to the p.v. Parasite destruction had several features in common with lysosomal and leukocyte damage induced by the esters, i.e., inactivity of D-amino acid esters, a marked pH dependence and increased killing after ester pulses at lower temperatures. Killing depended on the amino acid and on the ester substitution. The most active of the methyl esters assayed was that of leucine, followed by those of tryptophan, glutamic acid, methionine, phenylalanine, and tyrosine. Methyl esters of seven other amino acids were inactive when tested at up to 10 mM concentrations. Among leucine esters studied, benzyl ester was sixfold more active than the methyl homolog. The dipeptide L-leu-leu-OMe produced 90% cure at 0.08 mM concentrations. Leishmanicidal activity could be related to penetration of the parasites by the esters or to toxic ester hydrolysis products released in the p.v. The first hypothesis is supported by the pH-dependent destruction of isolated amastigotes by the esters. Furthermore, relatively high concentrations of L-leucine, methanol, or benzyl alcohol were not demonstrably toxic to the amastigotes. We postulate that ester concentrations sufficient to damage the intracellular amastigotes may be obtained within the p.v. after exposure of infected macrophages to the esters. Esters preferentially hydrolyzed by parasite enzymes may be expected to be leishmanicidal, but less damaging to the host.

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Leishmania amastigotes, obligatory parasites of macrophages, lodge and multiply within long-lived phagolysosomelike "parasitophorous vacuoles" (PV). The glycoprotein horseradish peroxidase (HRP) was shown, by light and electron microscopic cytochemistry, to enter the PVs of rat in vitro-derived bone marrow macrophages infected with Leishmania mexicana amazonensis. Uptake was obtained both in preinfected macrophages incubated with HRP and in macrophages pulsed with HRP, infected, and further incubated in ligand-free medium. Peroxidase positive and negative PVs could coexist in the same macrophages. Infected macrophages commonly displayed fewer labeled secondary lysosomes than noninfected cells. Lactoperoxidase (LP) was also shown, by light microscopy, to enter the PVs of rat macrophages. Uptake of HRP and of LP was blocked by mannan, supporting the mannose receptor mediated recognition of these ligands. Transfer of HRP to PVs was much less efficient in resident mouse peritoneal macrophages, even at 50 X higher ligand concentrations. Such macrophages expressed negligible mannose receptor function. The efficient mannan-inhibitable uptake of HRP by rat marrow macrophages was confirmed biochemically. Bulk HRP uptake in infected and noninfected cultures was found to be similar. Peroxidases should be useful in further studies of endocytosis by Leishmania-infected macrophages and in the development of lysosomotropic macrophage-targeted drug carriers.

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Leishmania amastigotes lodge and multiply within parasitophorous vacuoles, which can fuse with secondary lysosomes of the host macrophages. This study examines the effect of infection with amastigotes of L. mexicana amazonensis on the secondary lysosomes of mouse macrophage cultures. The cultures were stained for the activities of two lysosomal enzyme markers, acid phosphatase and arylsulfatase, and the light microscopic observations were supplemented by electron microscopy. Nearly all noninfected macrophages contained numerous stained secondary lysosomes. The number of such lysosomes was markedly reduced 24 h postinfection, and the reduction persisted for at least 10 days. Stained secondary lysosomes reappeared after the amastigotes were destroyed by exposure of the cultures to phenazine methosulfate or by placing them at 37.5 degrees C. The depletion of lysosomes shown by cytochemical methods may reflect a high rate of fusion of the lysosomes with the parasitophorous vacuoles, exceeding the rate of formation of new secondary lysosomes. Alternatively, the parasites may inhibit the synthesis of lysosomal hydrolases, or the assembly or formation of primary or secondary lysosomes.

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Phenazine methosulfate, a cationic electron carrier, inhibits the extracellular growth of promastigotes and the conversion of amastigotes into promastigote forms of Leishmania mexicana amazonensis. Growth inhibition and damage of extracellular parasites by PMS was counteracted by superoxide dismutase, a scavenger of the superoxide anion (O2-), and to a lesser extent, by catalase, a scavenger of hydrogen peroxide (H2O2). Inactivated dismutase and catalase were ineffective. Thus, damage of isolated L.m. amazonensis by phenazine methosulfate, involves the participation of O2- and H2O2. The role of the oxygen metabolites in the toxicity of phenazine methosulfate remains unknown. That O2- can damage the parasites is supported by the finding that superoxide dismutase also protected promastigotes from damage induced by oxygen intermediates generated by a xanthine-xanthine oxidase system. Killing of the parasites by crystal violet, a triphenylmethane, or basic blue 24, a phenothiazine, was not inhibited by superoxide dismutase.

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Different temperature requirements for intracellular growth of Leishmania spp. may account for the predominantly peripheral or visceral localization of the parasites in the vertebrate host. We show here that Leishmania mexicana amazonensis, an agent of cutaneous disease, is growth restricted in vitro at 37.5 C and viable at 34 C. This temperature sensitivity has been observed in both infected macrophage cultures and isolated amastigotes. Counts of the percentage of infected macrophages and of the number of amastigotes per infected cell documented that at 34 C the parasites progressively proliferated for at least 11 days. In contrast, no amastigotes were found in infected cells 48 to 72 hr after the cultures were shifted to the restrictive temperature of 37.5 C. This effect was reversible within the first 24 hr of temperature shift. Incorporation of [3H]uracil into parasite RNA was used to monitor the viability of intracellular and of isolated amastigotes. The trichloroacetic acid-precipitable incorporation of [3H]uracil by infected macrophages was markedly reduced as early as 6 hr after the monolayers were placed at 37.5 C. This decrease was also reversible within the first 24 hr at the restrictive temperature. In order to determine whether the restrictive temperature could directly affect the parasites, isolated amastigotes were incubated at either 34 C or 37.5 C and assayed for [3H]uracil incorporation. The results indicated that by 3 hr of incubation at the restrictive temperature the incorporation was significantly decreased and remained low for at least 9 hr.

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125I-labeled rat preputial gland beta-glucuronidase was shown by light and electron microscopic radioautography to accumulate within the parasitophorous vacuoles of in vitro derived bone marrow macrophages infected with Leishmania mexicana amazonensis. beta-glucuronidase uptake was mediated by the mannose receptor, since the penetration of the ligand was inhibited by mannan. Uptake was detected as soon as 4 h after incubation of infected cells with the ligand, and increased at 24 and 48 h. The label persisted in the vacuoles for at least 24 h after a 24-h pulse with the ligand, a finding compatible with the relatively long half-life of labeled beta-glucuronidase in normal macrophages. Parasitophorous vacuoles were also labeled in macrophages exposed to the ligand only before infection, indicating that secondary lysosomes containing the ligand fused with the parasitophorous vacuoles. Another mannosylated ligand, mannose-BSA, which, in contrast to beta-glucuronidase, is rapidly degraded in macrophage lysosomes, did not detectably accumulate in the vacuoles. The results support and extend information previously obtained with electron opaque tracers that emphasizes the phagolysosomal nature of Leishmania parasitophorous vacuoles. In addition, the results suggest that appropriate mannosylated molecules may be used as carriers for targeting of leishmanicidal drugs to the parasitophorous vacuoles of infected macrophages.

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