Search

Environmental factors such as diet, gut microbiota, and infections have proven to have a significant role in epigenetic modifications. It is known that epigenetic modifications may cause behavioral and neuronal changes observed in neurodevelopmental disabilities, including fragile X syndrome (FXS) and autism (ASD). Probiotics are live microorganisms that provide health benefits when consumed, and in some cases are shown to decrease the chance of developing neurological disorders. Here, we examined the epigenetic outcomes in offspring mice after feeding of a probiotic organism, Lactobacillus reuteri (L. reuteri), to pregnant mother animals. In this study, we tested a cohort of Western diet-fed descendant mice exhibiting a high frequency of behavioral features and lower FMRP protein expression similar to what is observed in FXS in humans (described in a companion manuscript in this same GENES special topic issue). By investigating 17,735 CpG sites spanning the whole mouse genome, we characterized the epigenetic profile in two cohorts of mice descended from mothers treated and non-treated with L. reuteri to determine the effect of prenatal probiotic exposure on the prevention of FXS-like symptoms. We found several genes involved in different neurological pathways being differentially methylated (p ≤ 0.05) between the cohorts. Among the key functions, synaptogenesis, neurogenesis, synaptic modulation, synaptic transmission, reelin signaling pathway, promotion of specification and maturation of neurons, and long-term potentiation were observed. The results of this study are relevant as they could lead to a better understanding of the pathways involved in these disorders, to novel therapeutics approaches, and to the identification of potential biomarkers for early detection of these conditions.

Myelination in the peripheral and central nervous systems is critical in regulating motor, sensory, and cognitive functions. As myelination occurs rapidly during early life, neonatal gut dysbiosis during early colonization can potentially alter proper myelination by dysregulating immune responses and neuronal differentiation. Despite common usage of antibiotics (Abx) in children, the impact of neonatal Abx-induced dysbiosis on the development of microbiota, gut, brain (MGB) axis, including myelination and behavior, is unknown. We hypothesized that neonatal Abx-induced dysbiosis dysregulates host-microbe interactions, impairing myelination in the brain, and altering the MGB axis. Neonatal C57BL/6 mice were orally gavaged daily with an Abx cocktail (neomycin, vancomycin, ampicillin) or water (vehicle) from postnatal day 7 (P7) until weaning (P23) to induce gut dysbiosis. Behavior (cognition; anxiety-like behavior), microbiota sequencing, and qPCR (ileum, colon, hippocampus and pre-frontal cortex [PFC]) were performed in adult mice (6–8 weeks). Neonatal Abx administration led to intestinal dysbiosis in adulthood, impaired intestinal physiology, coupled with perturbations of bacterial metabolites and behavioral alterations (cognitive deficits and anxiolytic behavior). Expression of myelin-related genes (Mag, Mog, Mbp, Mobp, Plp) and transcription factors (Sox10, Myrf) important for oligodendrocytes were significantly increased in the PFC region of Abx-treated mice. Increased myelination was confirmed by immunofluorescence imaging and western blot analysis, demonstrating increased expression of MBP, SOX10 and MYRF in neonatally Abx-treated mice compared to sham controls in adulthood. Finally, administration of the short chain fatty acid butyrate following completion of the Abx treatment restored intestinal physiology, behavior, and myelination impairments, suggesting a critical role for the gut microbiota in mediating these effects. Taken together, we identified a long-lasting impact of neonatal Abx administration on the MGB axis, specifically on myelin regulation in the PFC region, potentially contributing to impaired cognitive function and bacterial metabolites are effective in reversing this altered phenotype.

Diarrheal diseases are a leading cause of death in children under the age of 5 years worldwide. Repeated early-life exposures to diarrheal pathogens can result in comorbidities including stunted growth and cognitive deficits, suggesting an impairment in the microbiota-gut-brain (MGB) axis. Neonatal C57BL/6 mice were infected with enteropathogenic Escherichia coli (EPEC) (strain e2348/69; ΔescV [type III secretion system {T3SS} mutant]) or the vehicle (Luria-Bertani [LB] broth) via orogastric gavage at postnatal day 7 (P7). Behavior (novel-object recognition [NOR] task, light/dark [L/D] box, and open-field test [OFT]), intestinal physiology (Ussing chambers), and the gut microbiota (16S Illumina sequencing) were assessed in adulthood (6 to 8 weeks of age). Neonatal infection of mice with EPEC, but not the T3SS mutant, caused ileal inflammation in neonates and impaired recognition memory (NOR task) in adulthood. Cognitive impairments were coupled with increased neurogenesis (Ki67 and doublecortin immunostaining) and neuroinflammation (increased microglia activation [Iba1]) in adulthood. Intestinal pathophysiology in adult mice was characterized by increased secretory state (short-circuit current [I(sc)]) and permeability (conductance) (fluorescein isothiocyanate [FITC]-dextran flux) in the ileum and colon of neonatally EPEC-infected mice, along with increased expression of proinflammatory cytokines (Tnfα, Il12, and Il6) and pattern recognition receptors (Nod1/2 and Tlr2/4). Finally, neonatal EPEC infection caused significant dysbiosis of the gut microbiota, including decreased Firmicutes, in adulthood. Together, these findings demonstrate that infection in early life can significantly impair the MGB axis in adulthood.

Maternal microbial dysbiosis has been implicated in adverse postnatal health conditions in offspring, such as obesity, cancer, and neurological disorders. We observed that the progeny of mice fed a Westernized diet (WD) with low fiber and extra fat exhibited higher frequencies of stereotypy, hyperactivity, cranial features and lower FMRP protein expression, similar to what is typically observed in Fragile X Syndrome (FXS) in humans. We hypothesized that gut dysbiosis and inflammation during pregnancy influenced the prenatal uterine environment, leading to abnormal phenotypes in offspring. We found that oral in utero supplementation with a beneficial anti-inflammatory probiotic microbe, Lactobacillus reuteri, was sufficient to inhibit FXS-like phenotypes in offspring mice. Cytokine profiles in the pregnant WD females showed that their circulating levels of pro-inflammatory cytokine interleukin (Il)-17 were increased relative to matched gravid mice and to those given supplementary L. reuteri probiotic. To test our hypothesis of prenatal contributions to this neurodevelopmental phenotype, we performed Caesarian (C-section) births using dissimilar foster mothers to eliminate effects of maternal microbiota transferred during vaginal delivery or nursing after birth. We found that foster-reared offspring still displayed a high frequency of these FXS-like features, indicating significant in utero contributions. In contrast, matched foster-reared progeny of L. reuteri-treated mothers did not exhibit the FXS-like typical features, supporting a key role for microbiota during pregnancy. Our findings suggest that diet-induced dysbiosis in the prenatal uterine environment is strongly associated with the incidence of this neurological phenotype in progeny but can be alleviated by addressing gut dysbiosis through probiotic supplementation.

N-glycosylation is a ubiquitous posttranslational modification that affects protein structure and function, including those of the central nervous system. N-glycans attached to cell membrane proteins play crucial roles in all aspects of biology, including embryogenesis, development, cell–cell recognition and adhesion, and cell signaling and communication. Although brain function and behavior are known to be regulated by the N-glycosylation state of numerous cell surface glycoproteins, our current understanding of brain glycosylation is limited, and glycan variations associated with functional brain regions remain largely unknown. In this work, we used a well-established cell surface glycomic nanoLC-Chip-Q-TOF platform developed in our laboratory to characterize the N-glycome of membrane fractions enriched in cell surface glycoproteins obtained from specific functional brain areas. We report the cell membrane N-glycome of two major developmental divisions of mice brain with specific and distinctive functions, namely the forebrain and hindbrain. Region-specific glycan maps were obtained with ∼120 N-glycan compositions in each region, revealing significant differences in “brain-type” glycans involving high mannose, bisecting, and core and antenna fucosylated species. Additionally, the cell membrane N-glycome of three functional regions of the forebrain and hindbrain, the cerebral cortex, hippocampus, and cerebellum, was characterized. In total, 125 N-glycan compositions were identified, and their region-specific expression profiles were characterized. Over 70 N-glycans contributed to the differentiation of the cerebral cortex, hippocampus, and cerebellum N-glycome, including bisecting and branched glycans with varying degrees of core and antenna fucosylation and sialylation. This study presents a comprehensive spatial distribution of the cell-membrane enriched N-glycomes associated with five discrete anatomical and functional brain areas, providing evidence for the presence of a previously unknown brain glyco-architecture. The region-specific molecular glyco fingerprints identified here will enable a better understanding of the critical biological roles that N-glycans play in the specialized functional brain areas in health and disease., Graphical Abstract, Highlights • Cell membrane N-glycome maps of functional mouse brain areas were obtained. • Highly diverse N-glycome varies in a region-specific manner. • 75+ glycan motives discriminate region-specific cell membrane N-glycome., In Brief We have characterized the cell-membrane N-glycome of two major developmental divisions of the brain, the forebrain and hindbrain, and three functional derivatives from them, including the cerebral cortex, hippocampus, and cerebellum, revealing an extraordinary diversity of N-glycans expressed in a global and functional region-specific manner in the adult mouse. Furthermore, we identified +25 N-glycans able to differentiate the forebrain and hindbrain N-glycome. Additionally, over 35 N-glycans distinguished the cortex, hippocampus, and cerebellum N-glycomes and may serve as region-specific glycan biomarkers.

The gut microbiota and associated metabolites have emerged as potential modulators of pathophysiological changes in obesity and related metabolic disorders. Butyrate, a product of bacterial fermentation, has been shown to have beneficial effects in obesity and rodent models of diet-induced obesity. Here, we aimed to determine the beneficial effects of butyrate (as glycerol ester of butyrate monobutyrin, MB) supplementation on metabolic phenotype, intestinal permeability and inflammation, feeding behavior, and the gut microbiota in low-fat (LF)- and high-fat (HF)-fed mice. Two cohorts (separated by 2 weeks) of male C57BL/6J mice (n = 24 in each cohort, 6/group/cohort, 6 weeks old) were separated into four weight-matched groups and fed either a LF (10 % fat/kcal) or HF (45% fat/kcal) with or without supplementation of MB (LF/MB or HF/MB) at 0.25% (w/v) in drinking water for 6 weeks. Metabolic phenotypes (body weight and adiposity), intestinal inflammation, feeding behavior, and fecal microbiome and metabolites were measured. Despite identical genetic and experimental conditions, we found marked differences between cohorts in the response (body weight gain, adiposity, and intestinal permeability) to HF-diet and MB. Notably, the composition of the gut microbiota was significantly different between cohorts, characterized by lower species richness and differential abundance of a large number of taxa, including subtaxa from five phyla, including increased abundance of the genera Bacteroides, Proteobacteria, and Parasutterella in cohort 2 compared to cohort 1. These differences may have contributed to the differential response in intestinal permeability to the HF diet in cohort 2. MB supplementation had no significant effect on metabolic phenotype, but there was a trend to protect from HF-induced impairments in intestinal barrier function in cohort 1 and in sensitivity to cholecystokinin (CCK) in both cohorts. These data support the concept that microbiota composition may have a crucial effect on metabolic responses of a host to dietary interventions and highlight the importance of taking steps to ensure reproducibility in rodent studies.

Key points •Nucleotide binding oligomerization domain (Nod)-like receptors regulate cognition, anxiety and hypothalamic-pituitary-adrenal axis activation. •Nod-like receptors regulate central and peripheral serotonergic biology. •Nod-like receptors are important for maintenance of gastrointestinal physiology. •Intestinal epithelial cell expression of Nod1 receptors regulate behaviour. Abstract Gut-brain axis signalling is critical for maintaining health and homeostasis. Stressful life events can impact gut-brain signalling, leading to altered mood, cognition and intestinal dysfunction. In the present study, we identified nucleotide binding oligomerization domain (Nod)-like receptors (NLR), Nod1 and Nod2, as novel regulators for gut-brain signalling. NLR are innate immune pattern recognition receptors expressed in the gut and brain, and are important in the regulation of gastrointestinal physiology. We found that mice deficient in both Nod1 and Nod2 (NodDKO) demonstrate signs of stress-induced anxiety, cognitive impairment and depression in the context of a hyperactive hypothalamic-pituitary-adrenal axis. These deficits were coupled with impairments in the serotonergic pathway in the brain, decreased hippocampal cell proliferation and immature neurons, as well as reduced neural activation. In addition, NodDKO mice had increased gastrointestinal permeability and altered serotonin signalling in the gut following exposure to acute stress. Administration of the selective serotonin reuptake inhibitor, fluoxetine, abrogated behavioural impairments and restored serotonin signalling. We also identified that intestinal epithelial cell-specific deletion of Nod1 (VilCre+ Nod1f/f ), but not Nod2, increased susceptibility to stress-induced anxiety-like behaviour and cognitive impairment following exposure to stress. Together, these data suggest that intestinal epithelial NLR are novel modulators of gut-brain communication and may serve as potential novel therapeutic targets for the treatment of gut-brain disorders.

The peripheral nervous system is an active participant in immune responses capable of blocking aberrant activation of a variety of immune cells. As one of these neuro-immune circuits, the cholinergic anti-inflammatory pathway has been well established to reduce the severity of several immunopathologies. While the activation of this pathway by vagal nerve stimulation requires sympathetic innervation of the spleen, the neuro-immune circuitry remains highly controversial. Neuro-immune pathways in other lymphoid tissues such as mesenteric lymph nodes (MLN) that are critical to the surveillance of the small intestine and proximal colon have not been assessed. Using conditionally expressed Channelrhodopsin, selective stimulation of sympathetic post-ganglionic neurons in the superior mesenteric ganglion (SMG) prevented macrophage activation and LPS-induced TNFα production in the spleen and MLN, but not in the inguinal LN. Site selective stimulation of the SMG induced the release of norepinephrine, resulting in β2AR dependent acetylcholine release in the MLN and spleen. VNS-evoked release of norepinephrine and acetylcholine in the MLN and spleen was significantly reduced using selective optogenetic blockade applied at the SMG. Additionally, this optogenetic blockade restored LPS-induced TNFα production, despite VNS. These studies identify the superior mesenteric ganglion as a critical node in a neuro-immune circuit that can inhibit immune function in the MLN and the spleen.

Glycosphingolipids (GSLs) are found in cellular membranes of most organisms and play important roles in cell-cell recognition, signaling, growth, and adhesion, among others. A method based on nanoflow high performance liquid chromatography-chip-quadrupole-time-of-flight mass spectrometry (nanoHPLC Chip-Q-TOF MS) was applied towards identifying and quantifying intact GSLs from a variety of samples, including cultured cell lines and animal tissue. The method provides the composition and sequence of the glycan, as well as variations in the ceramide portion of the GSL. It was used to profile the changes in the glycolipidome of Caco-2 cells as they undergo differentiation. A total of 226 unique GSLs were found among Caco-2 samples from five differentiation time-points. The method provided a comprehensive glycolipidomic profile of a cell during differentiation to yield the dynamic variation of intact GSL structures.

The regulation of mucosal immune function is critical to host protection from enteric pathogens but is incompletely understood. The nervous system and the neurotransmitter acetylcholine play an integral part in host defense against enteric bacterial pathogens. Here we report that acetylcholine producing-T-cells, as a non-neuronal source of ACh, were recruited to the colon during infection with the mouse pathogen Citrobacter rodentium. These ChAT+ T-cells did not exclusively belong to one Th subset and were able to produce IFNγ, IL-17A and IL-22. To interrogate the possible protective effect of acetylcholine released from these cells during enteric infection, T-cells were rendered deficient in their ability to produce acetylcholine through a conditional gene knockout approach. Significantly increased C. rodentium burden was observed in the colon from conditional KO (cKO) compared to WT mice at 10 days post-infection. This increased bacterial burden in cKO mice was associated with increased expression of the cytokines IL-1β, IL-6, and TNFα, but without significant changes in T-cell and ILC associated IL-17A, IL-22, and IFNγ, or epithelial expression of antimicrobial peptides, compared to WT mice. Despite the increased expression of pro-inflammatory cytokines during C. rodentium infection, inducible nitric oxide synthase (Nos2) expression was significantly reduced in intestinal epithelial cells of ChAT T-cell cKO mice 10 days post-infection. Additionally, a cholinergic agonist enhanced IFNγ-induced Nos2 expression in intestinal epithelial cell in vitro. These findings demonstrated that acetylcholine, produced by specialized T-cells that are recruited during C. rodentium infection, are a key mediator in host-microbe interactions and mucosal defenses., Author summary The nervous system is an active contributor to the regulation of immune responses. Prior studies have identified a unique CD4+ T-cell population that can relay signals from the sympathetic nervous system. These specialized T-cells express the enzyme choline acetyltransferase (ChAT) and produce acetylcholine (ACh). Release of ACh in response to neurotransmitters from the sympathetic innervation was previously shown to aberrant immune cell activation, reducing mortality during septic shock. Also, these CD4+ ChAT+ T-cells were previously found to control host-commensal interactions in naïve mice, but their role during enteric bacterial infection was unknown. Here we demonstrate that infection with C. rodentium induces ChAT+ T-cell recruitment and that expression of ChAT by this T-cell population significantly augments host defenses. These data support a diverse and expanding role of ACh in host immune responses.

Summary Newborns are colonized with an intestinal microbiota shortly after birth, but the factors governing the retention and abundance of specific microbial lineages are unknown. Nursing infants consume human milk oligosaccharides (HMOs) that pass undigested to the distal gut, where they may be digested by microbes. We determined that the prominent neonate gut residents, Bacteroides thetaiotaomicron and Bacteroides fragilis , induce the same genes during HMO consumption that are used to harvest host mucus glycans, which are structurally similar to HMOs. Lacto- N -neotetraose, a specific HMO component, selects for HMO-adapted species such as Bifidobacterium infantis , which cannot use mucus, and provides a selective advantage to B. infantis in vivo when biassociated with B. thetaiotaomicron in the gnotobiotic mouse gut. This indicates that the complex oligosaccharide mixture within HMOs attracts both mutualistic mucus-adapted species and HMO-adapted bifidobacteria to the infant intestine that likely facilitate both milk and future solid food digestion.

Given the biological importance of glycosylation on proteins, the identification of protein glycosylation sites is integral to understanding broader biological structure and function. Unfortunately, the determination of the microheterogeneity at the site of glycosylation still remains a significant challenge. Nanoflow liquid chromatography with tandem mass spectrometry provides both separation of glycopep- tides and the ability to determine glycan composition and site-specific glycosylation. However, because of the size of glycopeptides, they are not often amenable to tandem MS. In this work, proteins are digested with multiple proteases to produce glycopeptides that are of suitable size for tandem MS analysis. The conditions for collision-induced dissociation are optimized to obtain diagnostic ions that maximize glycan and peptide information. The method is applied to glycoproteins with contrasting glycans and multiple sites of glycosylation and identifies multiple glycan compositions at each individual glycosylation site. This method provides an important improvement in the routine determination of glycan microheterogeneity by mass spectrometry.

With the aim to study proteinases released to the culture medium duringTrypanosoma cruzimetacyclogenesis, the presence of cysteine proteinases (CPs) was analysed in culture supernatants obtained throughout the differentiation induced by stimulation of epimastigotes withTriatoma infestanshindgut homogenate. In SDS-gelatin containing gels, an important endopeptidase activity with apparent molecular weight range between 97 and 116 kDa was encountered at pH 6, which was abolished by the specific cysteine proteinase inhibitor E-64 and TLCK, but not by pepstatin, 1,10 phenantroline or PMSF. This novel CP, namedTcCPmet, showed affinity to cystatin-Sepharose, denoting its thiol-proteinase character as well as to ConA-Sepharose, indicating it contains N-linked oligosaccharides. However, it presented a different elution pattern on ConA-Sepharose than cruzipain and, in addition, it was not recognized by anti-cruzipain serum, facts that strongly suggest the different nature of both CPs. Moroever, evidence is presented indicating thatTcCPmet was able to hydrolyse the same chromogenic peptides as cruzipain at optimal alkaline pH values, although with a different order of effectiveness. Our results indicate the presence of a novel CP secreted by metacyclic trypomastigotes and reinforces the important role of these enzymes in metacyclogenesis.

The present paper reports the partial characterization of a subset of atypical cruzipain molecules which do not bind to Concanavalin A-Sepharose column. They are present in different strains of epimastigote forms of Trypanosoma cruzi and represent a 2-4% of total cruzipain. They were purified by affinity chromatography on Cystatin-Sepharose, recognized by the polyclonal anti-cruzipain serum, and their activity in gelatin-containing gels was completely abolished by E-64, TLCK, leupeptin, and aprotinin but not by PMSF, pepstatin A, EDTA or 1,10-phenantroline. These cysteine proteinases, as well as cruzipain showed to be endoproteinases able to hydrolize azocasein, hemoglobin, and bovine serum albumin at acidic pHs. However, evidences are presented indicating that this subset of cruzipain isoforms were also able to use the same blocked chromogenic peptidyl substrates than cruzipain at similar optimal alkaline pH values although with a different order of preference. Moreover, they showed a different oligosaccharide pattern after enzymatic treatment by high pH anion exchange chromatography, suggesting that this structural difference may account for the atypical behaviour in the lectin column.

Trypanosoma cruzi, the agent of Chagas disease contains a major cysteine proteinase, cruzipain (Cz), with an unusual carboxyl-terminal extension (C-T). We have previously reported the presence of sulfate groups in the N-linked oligosaccharide chains of this domain. In order to evaluate the immune responses to sulfated moieties on Cz, BALB/c mice were immunized with purified Cz and C-T prior and after desulfation treatment. The humoral immune response to sulfates on Cz or C-T was mainly IgG2b. Interestingly, the abolishment of IgG2b reactivity when desulfated antigens were used as immunogens demonstrates that esterified sulfate groups are absolutely required for eliciting IgG2b response to Cz. Sera from chronically T. cruzi-infected subjects with mild disease displayed higher levels of total IgG and IgG2 antibodies specific for sulfated epitopes compared with those in more severe forms of the disease. A significant reduction of C-T-specific delayed-type hypersensitivity reaction in C-T-immunized mice was observed when desulfated C-T was challenged, suggesting the involvement of sulfate groups in the generation of memory T-cell responses. Moreover, immunization with C-T in the absence of infection elicited ultrastructural abnormalities in heart tissue. Surprisingly, hearts from sulfate-depleted C-T-immunized mice did not present pathological alterations. This is the first report showing that sulfate-bearing glycoproteins from trypanosomatids are able to elicit specific humoral and cellular immune responses and appeared to be involved in the generation of heart tissue damage. These results represent a further step in the understanding of the role of Cz in the course of T. cruzi infection.

Trypanosoma cruzi, the parasitic protozoan that causes Chagas disease, contains a major cysteine proteinase, cruzipain. This lysosomal enzyme bears an unusual C-terminal extension that contains a number of post-translational modifications, and most antibodies in natural and experimental infections are directed against it. In this report we took advantage of UV-MALDI-TOF mass spectrometry in conjunction with peptide N-glycosidase F deglycosylation and high performance anion exchange chromatography analysis to address the structure of the N-linked oligosaccharides present in this domain. The UV-MALDI-TOF MS analysis in the negative-ion mode, using nor-harmane as matrix, allowed us to determine a new striking feature in cruzipain: sulfated high-mannose type oligosaccharides. Sulfated GlcNAc2Man3 to GlcNAc2Man9 species were identified. In accordance, after chemical or enzymatic desulfation, the corresponding signals disappeared. In addition, by UV-MALDI-TOF MS analysis (a) a main population of high-mannose type oligosaccharides was shown in the positive-ion mode, (b) lactosaminic glycans were also identified, among them, structures corresponding to monosialylated species were detected, and (c) as an interesting fact a fucosylated oligosaccharide was also detected. The presence of the deoxy sugar was further confirmed by high performance anion exchange chromatography. In conclusion, the total number of oligosaccharides occurring in cruzipain was shown to be much higher than previous estimates. This constitutes the first report on the presence of sulfated glycoproteins in Trypanosomatids.

This article has been retracted at the request of the authors and editor. Reason: This article was published previously in Molecular and Biochemical Parasitology, Vol. 126, issue 2, pages 293–296, February 2003. The retraction is due to dual publication and does not in any way imply any faults in the manuscript.

Plasmatic levels of pregnancy zone protein (PZP) increase in children with acute Chagas disease. PZP, as well as alpha2-macroglobulin (alpha2-M), are able to interact with Trypanosoma cruzi proteinases. The interaction of alpha2-M and PZP with cruzipain, the major cysteine proteinase of T. cruzi, was investigated. Several molecular changes on both alpha-M inhibitors under reaction with cruzipain were found. PAGE analysis showed: (i) formation of complexes of intermediate mobility and tetramerization of native alpha2-M and PZP, respectively; (ii) limited proteolysis of bait region in alpha2-M and PZP, and (iii) covalent binding of cruzipain to PZP and alpha2-M. Conformational and structural changes experimented by alpha-Ms correlate with modifications of the enzyme electrophoretic mobility and activity. Cruzipain-alpha-M complexes were also detected by gelatin SDS-PAGE and immunoblotting using polyclonal anti-cruzipain antibodies. Concomitantly, alpha2-M and PZP impaired the activity of cruzipain towards Bz-Pro-Phe-Arg-pNA substrate. In addition, alpha-Ms were able to form covalent complexes with membrane isoforms of cysteine proteinases cross-reacting with cruzipain. The present study suggests that both human alpha-macroglobulin inhibitors could prevent or minimize harmful action of cruzipain on host's molecules and hypothetically regulate parasite functions controlled by cruzipain.