Lrp5 is not required for the proliferative response of osteoblasts to strain but regulates proliferation and apoptosis in a cell autonomous manner

Although Lrp5 is known to be an important contributor to the mechanisms regulating bone mass, its precise role remains unclear. The aim of this study was to establish whether mutations in Lrp5 are associated with differences in the growth and/or apoptosis of osteoblast-like cells and their proliferative response to mechanical strain in vitro. Primary osteoblast-like cells were derived from cortical bone of adult mice lacking functional Lrp5 (Lrp5(-/-)), those heterozygous for the human G171V High Bone Mass (HBM) mutation (LRP5(G171V)) and their WT littermates (WT(Lrp5), WT(HBM)). Osteoblast proliferation over time was significantly higher in cultures of cells from LRP5(G171V) mice compared to their WT(HBM) littermates, and lower in Lrp5(-/-) cells. Cells from female LRP5(G171V) mice grew more rapidly than those from males, whereas cells from female Lrp5(-/-) mice grew more slowly than those from males. Apoptosis induced by serum withdrawal was significantly higher in cultures from Lrp5(-/-) mice than in those from WT(HBM) or LRP5(G171V) mice. Exposure to a single short period of dynamic mechanical strain was associated with a significant increase in cell number but this response was unaffected by genotype which also did not change the 'threshold' at which cells responded to strain. In conclusion, the data presented here suggest that Lrp5 loss and gain of function mutations result in cell-autonomous alterations in osteoblast proliferation and apoptosis but do not alter the proliferative response of osteoblasts to mechanical strain in vitro.     

Characterisation of membrane mimetics on a dual polarisation interferometer

Dual polarisation interferometry (DPI) has been used to characterise the formation of hybrid bilayer membranes (HBM) on a silicon-oxynitride surface. This technique allows the simultaneous determination of multiple physical properties of an HBM, as the HBM is being formed in a single experiment: mass, thickness in the z-direction (normal to the surface), tilt angle of the first layer and refractive index. Decanoic acid was covalently attached to an amine modified silicon-oxynitride sensor chip surface via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride condensation reaction. The decanoic acid layer was 0.92+/-0.12 nm thick, indicating a tilt angle of 57 degrees from surface normal, and possessed a mass of 1.05+/-0.10 ng mm(-2) and a refractive index (RI) of 1.450+/-0.020. Phospholipid vesicles made from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were injected over the fatty acid surface to form an HBM. The DPPC HBM was 4.32+/-0.68 nm thick, with a total mass of 3.18+/-0.60 ng mm(-2) and a RI of 1.404+/-0.007. The DMPC HBM was 2.12+/-0.34 nm thick, with a total mass of 2.25+/-0.51 ng mm(-2), and a RI of 1.435+/-0.007. DPI thus provides an insight into HBM formation and differences between the structural organisation of HBMs of different composition.     

Plasma membrane association of three classes of bacterial toxins is mediated by a basic-hydrophobic motif

Plasma membrane targeting is essential for the proper function of many bacterial toxins. A conserved fourhelical bundle membrane localization domain (4HBM) was recently identified within three diverse families of toxins: clostridial glucosylating toxins, MARTX toxins and Pasteurella multocida-like toxins. When expressed in tissue culture cells or in yeast, GFP fusions to at least one 4HBM from each toxin family show significant peripheral membrane localization but with differing profiles. Both in vivo expression and in vitro binding studies reveal that the ability of these domains to localize to the plasma membrane and bind negatively charged phospholipids requires a basic-hydrophobic motif formed by the L1 and L3 loops. The different binding capacity of each 4HBM is defined by the hydrophobicity of an exposed residue within the motif. This study establishes that bacterial effectors utilize a normal host cell mechanism to locate the plasma membrane where they can then access their intracellular targets.     

Substitution of the heme binding module in hemoglobin alpha- and beta-subunits. Implication for different regulation mechanisms of the heme proximal structure between hemoglobin and myoglobin

In our previous work, we demonstrated that the replacement of the "heme binding module," a segment from F1 to G5 site, in myoglobin with that of hemoglobin alpha-subunit converted the heme proximal structure of myoglobin into the alpha-subunit type (Inaba, K., Ishimori, K. and Morishima, I. (1998) J. Mol. Biol. 283, 311-327). To further examine the structural regulation by the heme binding module in hemoglobin, we synthesized the betaalpha(HBM)-subunit, in which the heme binding module (HBM) of hemoglobin beta-subunit was replaced by that of hemoglobin alpha-subunit. Based on the gel chromatography, the betaalpha(HBM)-subunit was preferentially associated with the alpha-subunit to form a heterotetramer, alpha(2)[betaalpha(HBM)(2)], just as is native beta-subunit. Deoxy-alpha(2)[betaalpha(HBM)(2)] tetramer exhibited the hyperfine-shifted NMR resonance from the proximal histidyl N(delta)H proton and the resonance Raman band from the Fe-His vibrational mode at the same positions as native hemoglobin. Also, NMR spectra of carbonmonoxy and cyanomet alpha(2)[betaalpha(HBM)(2)] tetramer were quite similar to those of native hemoglobin. Consequently, the heme environmental structure of the betaalpha(HBM)-subunit in tetrameric alpha(2)[betaalpha(HBM)(2)] was similar to that of the beta-subunit in native tetrameric Hb A, and the structural conversion by the module substitution was not clear in the hemoglobin subunits. The contrastive structural effects of the module substitution on myoglobin and hemoglobin subunits strongly suggest different regulation mechanisms of the heme proximal structure between these two globins. Whereas the heme proximal structure of monomeric myoglobin is simply determined by the amino acid sequence of the heme binding module, that of tetrameric hemoglobin appears to be closely coupled to the subunit interactions.     

Lactoferrin effects on phagocytic cell function. I. Increased uptake and killing of an intracellular parasite by murine macrophages and human monocytes

Mouse peritoneal macrophages (MPM) or human blood monocytes (HBM) co-cultured with intracellular (amastigote; AMA) forms of Trypanosoma cruzi in the presence of human lactoferrin (LF) took up greater numbers of organisms than in the absence of LF; the proportion of phagocytes taking up AMA was also significantly increased. Pretreatment of either MPM or AMA with LF also enhanced cell-parasite association. By immunofluorescence, HBM, MPM, and AMA were found to bind LF. By using 125I-labeled LF, each AMA was determined to have an average 1.1 X 10(6) surface receptors for LF. The enhancing effect of LF on cell-parasite association was inhibited when either rabbit anti-LF IgG or alpha-methyl mannoside (alpha-MM) was present during the incubation of MPM or AMA with LF, or when AMA pretreated with LF were then incubated with either of the LF blocking agents. Although these findings seemed to suggest that LF increased MPM-AMA association by bridging these cells, the LF effect was not inhibited when MPM pretreated with LF were subsequently incubated with either alpha-MM or anti-LF. Furthermore, LF stimulated phagocytosis, as denoted by a significant increase in latex particle uptake after LF treatment of MPM. The intracellular killing capacity of HBM or MPM was also stimulated by LF and was denoted by increased AMA destruction after LF treatments. The possibility that LF only appeared to increase the rate of AMA killing by simply promoting the engulfment of greater numbers of AMA that would then be destroyed intracellularly seemed unlikely because untreated MPM that had already taken up untreated AMA killed greater numbers of AMA when they were subsequently incubated with LF. The results of experiments with scavengers of oxygen reduction intermediates and of nitroblue tetrazolium reduction tests indicated that H2O2, O2- and 1O2 were involved in the killing of AMA by LF-treated MPM. These results suggest that LF, a glycoprotein secreted by neutrophils in greater than normal amounts during inflammation, may contribute to macrophage clearance of AMA released from infected host cells.     

Genetic Analysis of High Bone Mass Cases from the BARCOS Cohort of Spanish Postmenopausal Women

The aims of the study were to establish the prevalence of high bone mass (HBM) in a cohort of Spanish postmenopausal women (BARCOS) and to assess the contribution of LRP5 and DKK1 mutations and of common bone mineral density (BMD) variants to a HBM phenotype. Furthermore, we describe the expression of several osteoblast-specific and Wnt-pathway genes in primary osteoblasts from two HBM cases. A 0.6% of individuals (10/1600) displayed Z-scores in the HBM range (sum Z-score >4). While no mutation in the relevant exons of LRP5 was detected, a rare missense change in DKK1 was found (p.Y74F), which cosegregated with the phenotype in a small pedigree. Fifty-five BMD SNPs from Estrada et al. [NatGenet 44:491-501,2012] were genotyped in the HBM cases to obtain risk scores for each individual. In this small group of samples, Z-scores were found inversely related to risk scores, suggestive of a polygenic etiology. There was a single exception, which may be explained by a rare penetrant genetic variant, counterbalancing the additive effect of the risk alleles. The expression analysis in primary osteoblasts from two HBM cases and five controls suggested that IL6R, DLX3, TWIST1 and PPARG are negatively related to Z-score. One HBM case presented with high levels of RUNX2, while the other displayed very low SOX6. In conclusion, we provide evidence of lack of LRP5 mutations and of a putative HBM-causing mutation in DKK1. Additionally, we present SNP genotyping and expression results that suggest additive effects of several genes for HBM.     

Systematic microcarrier screening and agitated culture conditions improves human mesenchymal stem cell yield in bioreactors

Production of human mesenchymal stem cells for allogeneic cell therapies requires scalable, cost‐effective manufacturing processes. Microcarriers enable the culture of anchorage‐dependent cells in stirred‐tank bioreactors. However, no robust, transferable methodology for microcarrier selection exists, with studies providing little or no reason explaining why a microcarrier was employed. We systematically evaluated 13 microcarriers for human bone marrow‐derived MSC (hBM‐MSCs) expansion from three donors to establish a reproducible and transferable methodology for microcarrier selection. Monolayer studies demonstrated input cell line variability with respect to growth kinetics and metabolite flux. HBM‐MSC1 underwent more cumulative population doublings over three passages in comparison to hBM‐MSC2 and hBM‐MSC3. In 100 mL spinner flasks, agitated conditions were significantly better than static conditions, irrespective of donor, and relative microcarrier performance was identical where the same microcarriers outperformed others with respect to growth kinetics and metabolite flux. Relative growth kinetics between donor cells on the microcarriers were the same as the monolayer study. Plastic microcarriers were selected as the optimal microcarrier for hBM‐MSC expansion. HBM‐MSCs were successfully harvested and characterised, demonstrating hBM‐MSC immunophenotype and differentiation capacity. This approach provides a systematic method for microcarrier selection, and the findings identify potentially significant bioprocessing implications for microcarrier‐based allogeneic cell therapy manufacture.     

Divergent effects of zinc depletion in brain vs non-brain endothelial cells

Dietary zinc deficiency is common in developing as well as developed countries. Endothelial cells (EC) lining the inner surface of peripheral blood vessels are sensitive to zinc deficiency and lose structural integrity when exposed to culture media low in zinc or to zinc chelators. In contrast, we demonstrate here that human brain microvascular EC (HBMEC), which constitute the blood-brain barrier (BBB), resist zinc depletion and respond by enhancing their barrier function. This response was specific for HBMEC and did not occur in non-brain EC, such as human umbilical vein endothelial cells, human aortic endothelial cells, and human iliac vein endothelial cells. Our results suggest the presence of specific mechanisms to counteract zinc deficiency at the BBB, likely involving HBMEC junctional complexes. Understanding the mechanisms involved in this unique response might provide means to modulate the BBB dysfunction associated with neurological disorders such as stroke, multiple sclerosis, and Alzheimer's disease.     

Linkage of a Gene Causing High Bone Mass to Human Chromosome 11 (11q12-13)

The purpose of this paper is to report the linkage of a genetic locus (designated "HBM") in the human genome to a phenotype of very high spinal bone density, using a single extended pedigree. We measured spinal bone-mineral density, spinal Z(BMD), and collected blood from 22 members of this kindred. DNA was genotyped on an Applied Biosystems model 377 (ABI PRISM Linkage Mapping Sets; Perkin Elmer Applied Biosystems), by use of fluorescence-based marker sets that included 345 markers. Both two-point and multipoint linkage analyses were performed, by use of affected/unaffected and quantitative-trait models. Spinal Z(BMD) for affected individuals (N = 12) of the kindred was 5.54 +/- 1.40; and for unaffected individuals (N = 16) it was 0.41 +/- 0.81. The trait was present in affected individuals 18-86 years of age, suggesting that HBM influences peak bone mass. The only region of linkage was to a series of markers on chromosome 11 (11q12-13). The highest LOD score (5.21) obtained in two-point analysis, when a quantitative-trait model was used, was at D11S987. Multipoint analysis using a quantitative-trait model confirmed the linkage, with a LOD score of 5.74 near marker D11S987. HBM demonstrates the utility of spinal Z(BMD) as a quantitative bone phenotype that can be used for linkage analysis. Osteoporosis pseudoglioma syndrome also has been mapped to this region of chromosome 11. Identification of the causal gene for both traits will be required for determination of whether a single gene with different alleles that determine a wide range of peak bone densities exists in this region.     

Linkage studies in Lenz microphthalmia.

Glucose-6-phosphate dehydrogenase (G6PD) phenotype studies were done on a black family with X-linked heredofamilial bilateral microphthalmia (HBM). Three crossovers and three non-crossovers were detected in three informative matings of four generations yielding a recombination value of 0.5. These findings do not provide evidence for linkage between the G6PD and HBM loci, suggesting either that the G6PD and HBM loci are far apart on the X chromosome or that HBM in this family is inherited as an autosomal dominant male sex-limited trait.     

LRP5 mutations linked to high bone mass diseases cause reduced LRP5 binding and inhibition by SOST

The low density lipoprotein (LDL) receptor-related protein 5 (LRP5) is a co-receptor for Wnt proteins and a major regulator in bone homeostasis. Human genetic studies have shown that recessive loss-of-function mutations in LRP5 are linked to osteoporosis, while on the contrary, dominant missense LRP5 mutations are associated with high bone mass (HBM) diseases. All LRP5 HBM mutations are clustered in a single region in the LRP5 extracellular domain and presumably result in elevated Wnt signaling in bone forming cells. Here we show that LRP5 HBM mutant proteins exhibit reduced binding to a secreted bone-specific LRP5 antagonist, SOST, and consequently are more refractory to inhibition by SOST. As loss-of-function mutations in the SOST gene are associated with Sclerosteosis, another disorder of excessive bone growth, our study suggests that the SOST-LRP5 antagonistic interaction plays a central role in bone mass regulation and may represent a nodal point for therapeutic intervention for osteoporosis and other bone diseases.     

Missense Mutations in LRP5 Associated with High Bone Mass Protect the Mouse Skeleton from Disuse- and Ovariectomy-Induced Osteopenia

The low density lipoprotein receptor-related protein-5 (LRP5), a co-receptor in the Wnt signaling pathway, modulates bone mass in humans and in mice. Lrp5 knock-out mice have severely impaired responsiveness to mechanical stimulation whereas Lrp5 gain-of-function knock-in and transgenic mice have enhanced responsiveness to mechanical stimulation. Those observations highlight the importance of Lrp5 protein in bone cell mechanotransduction. It is unclear if and how high bone mass-causing (HBM) point mutations in Lrp5 alter the bone-wasting effects of mechanical disuse. To address this issue we explored the skeletal effects of mechanical disuse using two models, tail suspension and Botulinum toxin-induced muscle paralysis, in two different Lrp5 HBM knock-in mouse models. A separate experiment employing estrogen withdrawal-induced bone loss by ovariectomy was also conducted as a control. Both disuse stimuli induced significant bone loss in WT mice, but Lrp5 A214V and G171V were partially or fully protected from the bone loss that normally results from disuse. Trabecular bone parameters among HBM mice were significantly affected by disuse in both models, but these data are consistent with DEXA data showing a failure to continue growing in HBM mice, rather than a loss of pre-existing bone. Ovariectomy in Lrp5 HBM mice resulted in similar protection from catabolism as was observed for the disuse experiments. In conclusion, the Lrp5 HBM alleles offer significant protection from the resorptive effects of disuse and from estrogen withdrawal, and consequently, present a potential mechanism to mimic with pharmaceutical intervention to protect against various bone-wasting stimuli.     

Injury Related Risk Behaviour: A Health Belief Model-Based Study of Primary School Students in a Safe Community in Shanghai

Aim

To explore the relationship between Health belief model (HBM) and children and adolescents'' unintentional injury risk behavior, to add some useful information for injury prevention.

Methodology

We investigated injury related health risk behavior and health belief status of students at primary schools grade 3 to 4, in a Safe Community, in Shanghai. Self-administered injury questionnaires were used to investigate risk behavior of students and HBM factors.

Principal Findings

The prevalence of risk behavior among students reported in this community was high. HBM scores showed differences between two groups of students classified by whether they had risk behavior or not. Self-efficacy was highly related with the status of socio-psychological behavior.

Significance

HBM has been widely used in explaining the disease-related behavior; however, it has been seldom used in injury-related behavior. The study demonstrated important relation of HBM to students'' injury issues, and HBM could explain injury related behavior as well, especially for traffic injury-related behavior. When developing injury prevention strategies, we can take it into account.     

A Mutation in the LDL Receptor–Related Protein 5 Gene Results in the Autosomal Dominant High–Bone-Mass Trait

Osteoporosis is a complex disease that affects >10 million people in the United States and results in 1.5 million fractures annually. In addition, the high prevalence of osteopenia (low bone mass) in the general population places a large number of people at risk for developing the disease. In an effort to identify genetic factors influencing bone density, we characterized a family that includes individuals who possess exceptionally dense bones but are otherwise phenotypically normal. This high–bone-mass trait (HBM) was originally localized by linkage analysis to chromosome 11q12-13. We refined the interval by extending the pedigree and genotyping additional markers. A systematic search for mutations that segregated with the HBM phenotype uncovered an amino acid change, in a predicted β-propeller module of the low-density lipoprotein receptor–related protein 5 (LRP5), that results in the HBM phenotype. During analysis of >1,000 individuals, this mutation was observed only in affected individuals from the HBM kindred. By use of in situ hybridization to rat tibia, expression of LRP5 was detected in areas of bone involved in remodeling. Our findings suggest that the HBM mutation confers a unique osteogenic activity in bone remodeling, and this understanding may facilitate the development of novel therapies for the treatment of osteoporosis.     

Active muscle response using feedback control of a finite element human arm model

Mathematical human body models (HBMs) are important research tools that are used to study the human response in car crash situations. Development of automotive safety systems requires the implementation of active muscle response in HBM, as novel safety systems also interact with vehicle occupants in the pre-crash phase. In this study, active muscle response was implemented using feedback control of a nonlinear muscle model in the right upper extremity of a finite element (FE) HBM. Hill-type line muscle elements were added, and the active and passive properties were assessed. Volunteer tests with low impact loading resulting in elbow flexion motions were performed. Simulations of posture maintenance in a gravity field and the volunteer tests were successfully conducted. It was concluded that feedback control of a nonlinear musculoskeletal model can be used to obtain posture maintenance and human-like reflexive responses in an FE HBM.     

Reduced affinity to and inhibition by DKK1 form a common mechanism by which high bone mass-associated missense mutations in LRP5 affect canonical Wnt signaling

The low-density-lipoprotein receptor-related protein 5 (LRP5), a coreceptor in the canonical Wnt signaling pathway, has been implicated in human disorders of low and high bone mass. Loss-of-function mutations cause the autosomal recessive osteoporosis-pseudoglioma syndrome, and heterozygous missense mutations in families segregating autosomal dominant high bone mass (HBM) phenotypes have been identified. We expressed seven different HBM-LRP5 missense mutations to delineate the mechanism by which they alter Wnt signaling. None of the mutations caused activation of the receptor in the absence of ligand. Each mutant receptor was able to reach the cell surface, albeit at differing amounts, and transduce exogenously supplied Wnt1 and Wnt3a signal. All HBM mutant proteins had reduced physical interaction with and reduced inhibition by DKK1. These data suggest that HBM mutant proteins can transit to the cell surface in sufficient quantity to transduce Wnt signal and that the likely mechanism for the HBM mutations' physiologic effects is via reduced affinity to and inhibition by DKK1.     

Inhibition of GABA shunt enzymes' activity by 4-hydroxybenzaldehyde derivatives

4-Hydroxybenzaldehyde (HBA) derivatives were examined as inhibitors for GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH). Investigation of structure-activity relation revealed that a carbonyl group or an amino group as well as a hydroxy group at the para position of the benzene ring are important for both enzymes' inhibition. HBA was shown to give competitive inhibition of GABA-T with respect to alpha-ketoglutarate and competitive inhibition of SSADH. 4-Hydroxybenzylamine (HBM) also showed the competitive inhibition on GABA-T with respect to GABA. In conclusion, the inhibitory effects of HBA and HBM on both enzymes could result from the similarity between both molecules and the two enzymes' substrates in structure, as well as the conjugative effect of the benzene ring. This suggested that the presence of the benzene ring may be accepted by the active site of both enzymes, HBA and HBM may be considered as lead compounds to design novel GABA-T inhibitors.     

Active muscle response using feedback control of a finite element human arm model

Mathematical human body models (HBMs) are important research tools that are used to study the human response in car crash situations. Development of automotive safety systems requires the implementation of active muscle response in HBM, as novel safety systems also interact with vehicle occupants in the pre-crash phase. In this study, active muscle response was implemented using feedback control of a nonlinear muscle model in the right upper extremity of a finite element (FE) HBM. Hill-type line muscle elements were added, and the active and passive properties were assessed. Volunteer tests with low impact loading resulting in elbow flexion motions were performed. Simulations of posture maintenance in a gravity field and the volunteer tests were successfully conducted. It was concluded that feedback control of a nonlinear musculoskeletal model can be used to obtain posture maintenance and human-like reflexive responses in an FE HBM.     

高体重布氏田鼠产仔数较多但繁殖输出及氧化损伤不受影响

体重可影响动物几乎所有的生物学变量。高体重双亲通常产生较大胎仔数。繁殖过程代价很高,可能伴随着氧化损伤。为比较双亲体重对繁殖期能量学特征、繁殖输出和氧化损伤的影响,检测了高体重组(雌性:51. 5 g ± 1. 6 g;雄性:60. 4 g ±2. 5 g)和低体重组(雌性:35. 5 g ± 1. 2 g;雄性:49. 6 g ± 2. 8 g)布氏田鼠繁殖过程中母体的体重和摄食量,分娩时的胎仔数和胎仔总重,及后代断乳时母体的身体成分、激素含量(血清瘦素和催乳素)和氧化损伤(血清丙二醛、蛋白质羰基和肝脏丙二醛含量)。结果显示:(1)高体重组妊娠末期的母体体重增量显著高于低体重组;(2)高体重组母体的出生胎仔数显著高于低体重组,但出生及断乳时的平均胎仔重、胎仔总重和母体繁殖期间的能量摄入无显著差异; (3)血清激素含量、身体成分、身体脂肪重量及氧化损伤指标均无组间差异。结果表明,高体重布氏田鼠可产生较多的出生胎仔数,但母体并不通过摄入更多的能量来保证其繁殖输出,对氧化损伤也没有影响。这些结果对于理解不同体重的动物在繁殖期间的能量策略以及繁殖与生存之间的权衡等生活史理论具有重要意义。     

Role of inflammatory cells in Chagas' disease. II. Interactions of mouse macrophages and human monocytes with intracellular forms of Trypanosoma cruzi: uptake and mechanism of destruction

In this study we examined the kinetics of interaction between mouse peritoneal macrophages (MPH) or human blood monocytes (HBM) with intracellular (amastigote [AMA]) forms of Trypanosoma cruzi. In electron microscopy studies, AMA were seen bound to the surface of unelicited MPH after 5 min of interaction, i.e., when the first observations were made. Internalization was visible after 8 min, and the AMA were never seen outside of phagocytic vacuoles. Signs of AMA damage were first seen after 4 hr. Amastigote disintegration was commonly observed 12 hr after their initial contact with MPH. Similar results were obtained with HBM. These kinetic patterns of AMA uptake and destruction were in agreement with the results of quantitative assays in which the number of AMA contained by 200 MPH and the percentage of infected MPH were measured. The extent of the release of 3H-labeled materials from MPH that had phagocytosed [3H]AMA was approximately 10, 90, and 99% of the total ingested radioactivity after 4, 12, and 24 hr of incubation, respectively. A comparison of the kinetic patterns of MPH interaction with noninvasive AMA and invasive trypomastigote (TRY) forms showed that, after internalization, both the percentage of AMA-containing MPH and the number of AMA per 200 MPH declined dramatically over a 70-hr incubation period, whereas the percentage of MPH infected by the TRY remained virtually constant and the number of organisms per 200 cells increased markedly. This contrast indicated that the AMA had been destroyed, whereas the TRY had managed to survive, transform into AMA, and multiply within MPH. AMA killing by MPH involved H2O2 but not other intermediates of oxygen reduction, because it was inhibited by catalase but not by scavengers of O2, OH ., and 1O2. AMA lost their viability when incubated with glucose and glucose oxidase, confirming their sensitivity to H2O2. Thus, MPH and HBM have the potential for participating in the clearance of T. cruzi AMA from chagasic tissue lesions.