Roberto Kretschmer

Inhibition of human monocyte locomotion by products of axenically grown E. histolytica

Summary The supernatant fluid of axenically grown E. histolytica inhibits chemotaxis, chemokinesis and random mobility of human mononuclear phagocytes (MP) as measured in Boyden chambers. Human polymorphonuclear phagocytes (PMN) locomotion is apparently unaffected. The factor was found in comparable amounts in the supernatant fluid of axenic cultures of four E. histolytica strains that differed in their human pathogenicity and virulence, as well as in two entamoebas non‐pathogenic for man. This dialysable and thermolabile MP‐locomotion inhibiting entamoeba product (EP) can be absorbed out by incubation with MP, but not with lymphocytes, while partial absorption was observed using PMN. The MP‐locomotion inhibitory effect of this EP was cancelled by inhibiting protein synthesis in the MP by means of cycloheximide. In vivo, this EP caused a delay in MP migration in Rebuck skin windows. The molecular weight of this EP lies between 478 and 765 by gel‐sieve chromatography. Our results suggest a direct effect upon the cytoskeletal and locomotive apparatus of the MP. This MP‐locomotion inhibiting EP could contribute to the paucity of the inflammatory reaction observed in the advanced stages of invasive amoebiasis and consequently also to the lack of scar tissue formation upon healing of amoebic lesions.

The monocyte locomotion inhibitory factor produced by Entamoeba histolytica inhibits induced nitric oxide production in human leukocytes

Abstract. The monocyte locomotion inhibitory factor, an anti-inflammatory pentapeptide produced by Entamoeba histolytica, inhibits the in vitro production of nitric oxide induced by cytokines (INF-γ, TNF-α) or PMA in human leukocytes. This can be added to the other previously reported functional effects of this factor, such as the inhibition of monocyte locomotion and the synthesis of reactive oxygen intermediates in both monocytes and neutrophils. The decreased nitric oxide production may interfere with the killing of amebas by neutrophils in the early invasive stages of amebiasis, when oxidative mechanisms are used [reactive oxygen and nitrogen intermediates either individually or synergistically via peroxynitrite (ONOO–)], and in the advanced stages, when both non-oxidative and oxidative (including nitric oxide) mechanisms are employed by macrophages. Diminished nitric oxide production by leukocytes may also contribute to the paucity of late inflammatory components in amebic abscess of the liver and other amebic lesions.

A novel anti-inflammatory oligopeptide produced by Entamoeba histolytica

The monocyte locomotion inhibitory factor (MLIF), a heat-stable oligopeptide found in the supernatant fluid of Entamoeba histolytica axenic cultures was isolated by ultra-filtration, gel-sieve chromatography and high powered liquid chromatography (HPLC), and its primary structure (Met-Gln-Cys-Asn-Ser) established by Edman sequencing and mass-spectrometry (MS). A synthetic peptide had the same selective anti-inflammatory features as the native material in comparable concentrations: in vitro inhibition of the locomotion in human peripheral blood monocytes, and of the respiratory burst in the same cells and in human neutrophil polymorphonuclear leucocytes; and in vivo depression of delayed hypersensitivity skin reactions to dinitrochlorobenzene in guinea pigs. This oligopeptide is apparently synthesized by the ameba as suggested by [(35)S]-Cys and Met incorporation, probably as part of a larger molecule, from which it is cleaved by proteolysis. The full sequence was not found in the 431 available E. histolytica protein sequences. The factor may contribute to the unexpected paucity of the late inflammatory reaction found in advanced invasive amebiasis and, perhaps in consequence, to the regeneration without scarring (restitutio ad integrum) of the affected organs that is observed following successful treatment of this disease

An anti‐inflammatory oligopeptide produced by Entamoeba histolytica down‐regulates the expression of pro‐inflammatory chemokines

Axenically grown Entamoeba histolytica produces a pentapeptide (Met‐Gln‐Cys‐Asn‐Ser) with anti‐inflammatory properties that, among others, inhibits the in vitro and in vivo locomotion of human monocytes, sparing polymorphonuclear leucocytes from this effect [hence the name originally given: Monocyte Locomotion Inhibitory Factor (MLIF)]. A synthetic construct of this peptide displays the same effects as the native material. We now added MLIF to resting and PMA‐stimulated cells of a human monocyte cell line and measured the effect upon mRNA and protein expression of pro‐inflammatory chemokines (RANTES, IP‐10, MIP‐1α, MIP‐1β, MCP‐1, IL‐8, I‐309 and lymphotactin) and the shared CC receptor repertoire. The constitutive expression of these chemokines and the CC receptors was unaffected, whereas induced expression of MIP‐1α, MIP‐1β, and I‐309, and that of the CCR1 receptor – all involved in monocyte chemotaxis – was significantly inhibited by MLIF. This suggests that the inhibition of monocyte functions by MLIF may not only be exerted directly on these cells, but also – and perhaps foremost – through a conglomerate down‐regulation of endogenous pro‐inflammatory chemokines.

Effect of the monocyte locomotion inhibitory factor (MLIF) produced by Entamoeba histolytica on the expression of cell adhesion molecules (CAMs) in the skin of guinea pigs.

Entamoeba histolytica synthesizes a heat-stable pentapeptide, originally identified by one of its effects on mononuclear phagocytes (MP) as a monocyte locomotion inhibitory factor (MLIF). In addition to several other in vitro measurable antiinflammatory actions, MLIF was also found in vivo to delay the arrival of the late mononuclear leukocyte component in Rebuck human skin windows (1), and to inhibit the 1-chloro-2-4-dinitrobenzene (DNCB) contact hypersensitivity skin reaction in guinea pigs, a predominantly late mononuclear leukocyte inflammatory animal model (2). To explore whether MLIF also affects the regulation of cell-adhesion molecules (CAMs), we chose to measure the monocyte integrin VLA-4 and its corresponding endothelial ligand, the Ig superfamily VCAM-1, an interaction couple essential for the rolling, adhesion, and eventual extravasation of monocytes (3,4) in the course of a DNCB contact hypersensitivity skin reaction in guinea pigs.

Immunization with a tetramer derivative of an anti‐inflammatory pentapeptide produced by Entamoeba histolytica protects gerbils (Meriones unguiculatus) against experimental amoebic abscess of the liver

Axenically grown Entamoeba histolytica produces a pentapeptide (Met‐Gln‐Cys‐Asn‐Ser) with several anti‐inflammatory properties, including the inhibition of human monocyte locomotion (Monocyte Locomotion Inhibitory Factor (MLIF)). A construct displays the same effects as the native material. It remains to be seen if MLIF is used, or even produced in vivo by the tissue‐invading parasite. If MLIF were to be relevant in invasive amoebiasis, immunizing against it could diminish this parasite advantage and prevent lesions. KLH‐linked MLIF mixed with Freunds adjuvant was too aggressive an immunizing material to answer this question. However, immunization with a tetramer of MLIF (but not a scrambled version of MLIF) around a lysine core (MLIF–MAPS), that displays increased antigenicity, yet lacks excessive innate immunity activation, completely protects gerbils against amoebic abscess of the liver caused by the intraportal injection of virulent E. histolytica. Liver abscesses caused by Listeria monocytogenes were not prevented. Invasive E. histolytica may produce the parent protein of MLIF in vivo, and if appropriately cleaved, it may play a role in invasive amoebiasis. MLIF may join new vaccination strategies against amoebiasis.

Cytokine expression in CD4+ cells exposed to the monocyte locomotion inhibitory factor produced by Entamoeba histolytica

Entamoeba histolytica produces monocyte locomotion inhibitory factor (MLIF), a pentapeptide with in vitro and in vivo anti-inflammatory properties. MLIF may interfere with leukocyte migration, disturbing the balance of pro- and anti-inflammatory cytokines secreted by CD4+ T lymphocytes. We evaluated the effect of MLIF on expression of pro- and anti-inflammatory cytokines in human CD4+ T lymphocytes. Regulatory cytokines [interleukin-1 beta (IL-1β), IL-2, interferon gamma (IFN-γ), IL-5, IL-6, and IL-10] were studied by enzyme-linked immunosorbent assay method in CD4+-cell supernatant fluids. Proinflammatory cytokines were produced per se by MLIF (IL-1β, IL-2, and IFN-γ) and also anti-inflammatory cytokines (IL-5, IL-6, and IL-10) with 1-phorbol-12 myristate-13 acetate + MLIF; the IL-1β, IFN-γ, IL-5 and IL-6 production was inhibited but not that of IL-10 which disclosed increase in its expression. MLIF disturbs the pro- and anti-inflammatory balance, and it induces inhibition of IL-1β (principal proinflammatory cytokine) and increases IL-10 (prototype of an anti-inflammatory cytokine).

HLA antigens associated to amoebic abscess of the liver in Mexican mestizos

Summary Worldwide prevalence of amoebiasis is estimated at 4 ± 108 cases/year, yet only one of about 300 individuals harbouring Entamoeba histolytica suffers tissue invasion and these cases are mostly concentrated in certain areas of Asia, Africa and Latin America. Patients with amoebic abscess of the liver (AAL) represent only a small fraction of that. These contrasting figures have been tentatively explained on the one hand through variations in sex, immunocompetence. nutritional and other socioeconomic features of the host, and on the other hand through differences in parasite virulence. In order to explore a possible association between the major histocompatibility complex (MHC) and AAL susceptibility, we studied the HLA profile in 31 Mexican mestizos with AAL and compared it to race and socioeconomically matched controls. Mexican mestizo patients with AAL revealed a significant increase in HLA‐Bw16 and HLA‐DR3 which could suggest an HLA‐related susceptibility to liver invasion by E. histolytica.

Serum IgD concentration in pregnant women.

Abstract IgD levels were determined in maternal sera (obtained at delivery) at different stages of gestation, and found to be low in premature deliveries but normal in term deliveries when compared to those of normal nonpregnant women. Maternal IgD levels at delivery revealed a significant exponential correlation with duration of pregnancy. These results are at variance with the results of other surveys. The discrepancy could be explained on ethnic or methodological grounds. No correlation was found between maternal IgD levels and the degree of HL-A disparity between mother and product. Specificities HL-A-2 and HL-A-13 in the mother revealed a discrete correlation with IgD levels at delivery.

Does the eosinophil have a protective role in amebiasis

While normal human eosinophils are destroyed in vitro by virulent Entamoeba histolytica, notwithstanding the presence of antibodies and complement, activated eosinophils promptly destroy the parasite although dying also at the end of the process. To study the possible in vivo participation of eosinophils in invasive amebiasis, we compared the induction of experimental amebic abscess of the liver (AAL) in gerbils (Meriones unguiculatus) previously made eosinophilic through Toxocara canis antigen injection and in normal control gerbils. After intraportal inoculation of 10(5) ameba trophozoites (6 and 24 hr), the ratio of gerbils with AAL, as well as the number and size of the microabscesses was comparable in eosinophilic and control gerbils. However, at 96 hr the number and size of the microabscesses were significantly smaller (p < 0.05) in eosinophilic gerbils. On the other hand the actuarial AAL survival curve up to 45 days post-amebic inoculation was significantly (p < 0.05) shifted to the right in controls. These results suggest that antigen-induced eosinophilia may exert a protective effect against AAL in gerbils.