Complete immunization against Trypanosoma cruzi verified in individual mice by complement-mediated lysis

Experimental systems to assay immunity against Trypanosoma cruzi usually demonstrate partial resistance without excluding the establishment of sub-patent infections in protected animals. To test whether Swiss mice immunized with attenuated parasites might develop complete resistance against virulent T. cruzi, experiments were performed involving challenge with low numbers of parasites, enhancement of local inflammation and the combination of natural and acquired resistance. Absence of infection was established after repeated negative parasitological tests (including xenodiagnosis and hemoculture), and lack of lytic antibody was tested by complement mediated lysis. Immunization with 10(7) attenuated epimastigotes conferred protection against the development of high levels of parasitemia after challenge with Tulahuen strain, but was unable to reduce the number of infected animals. However, when a strong, delayed-type hypersensitivity reaction was triggered at the site of infection by injecting a mixture of virulent and attenuated T. cruzi, a significant proportion of immunized animals remained totally free of virulent infection. The same result was obtained when the immunization experiment was performed in four month old Swiss mice, displaying a relatively high natural resistance and challenged with wild, vector-borne parasites. These experiments demonstrate that complete resistance against T. cruzi can be obtained in a significant proportion of animals, under conditions which replicate natural, vector delivered infection by the parasite.

Complement-mediated regulation of Trichomonas vaginalis infection in mice

Trichomonas vaginalis is a flagellated protozoan which causes trichomoniasis, a sexually transmitted disease of the human genitourinary tract, The importance of the alternative complement pathway in host defence against T. vaginalis was investigated in vitro. Kinetic studies utilising immunofixation following electrophoresis showed that both a strongly and weakly virulent strain of T, vaginalis activated murine serum C3. In vivo studies with congenic-resistant, C5-deficient, B10.D2/OSn- and C5-sufficient, B10.D2/nSn mice showed that the presence of C5 is a significant factor in the innate host resistance to primary infection with a strongly virulent, but not a weakly virulent trichomonad strain.

Lack of galactose-alpha-1,3-galactose expression on porcine endothelial cells prevents complement-induced lysis but not direct xenogeneic NK cytotoxicity

The galactose-alpha-1,3-galactose (alphaGal) carbohydrate epitope is expressed on porcine, but not human cells, and therefore represents a major target for preformed human anti-pig natural Abs (NAb). Based on results from pig-to-primate animal models, NAb binding to porcine endothelial cells will likely induce complement activation, lysis, and hyperacute rejection in pig-to-human xenotransplantation. Human NK cells may also contribute to innate immune responses against xenografts, either by direct recognition of activating molecules on target cells or by FcgammaRIII-mediated xenogeneic Ab-dependent cellular cytotoxicity (ADCC). The present study addressed the question as to whether the lack of alphaGal protects porcine endothelial cells from NAb/complement-induced lysis, direct xenogeneic NK lysis, NAb-dependent ADCC, and adhesion of human NK cells under shear stress. Homologous recombination, panning, and limiting dilution cloning were used to generate an alphaGal-negative porcine endothelial cell line, PED2*3.51. NAb/complement-induced xenogeneic lysis of PED2*3.51 was reduced by an average of 86% compared with the alphaGal-positive phenotype. PED2*3.51 resisted NK cell-mediated ADCC with a reduction of lysis ranging from 30 to 70%. However, direct xenogeneic lysis of PED2*3.51, mediated either by freshly isolated or IL-2-activated human NK cells or the NK cell line NK92, was not reduced. Furthermore, adhesion of IL-2-activated human NK cells did not rely on alphaGal expression. In conclusion, removal of alphaGal leads to a clear reduction in complement-induced lysis and ADCC, but does not resolve adhesion of NK cells and direct anti-porcine NK cytotoxicity, indicating that alphaGal is not a dominant target for direct human NK cytotoxicity against porcine cells.

Difference in susceptibility to lysis between clones of the Y strain of Trypanosoma cruzi

Three clones isolated from the Y strain of Trypanosoma cruzi - YP1, YP2 and YP3 - were adapted to in vitro cultivation in VERO cells. The recovery of the parasites from the Y strain and clone YP3 was similar after 24 hr of contact with cells (3.2% and 2.7%, respectively) and much lower than the recovery of clones YP1 and YP2 (56.7% and 60.0% of inoculum, respectively). After five days incubation, the ratio Trypomastigotes/Amastigotes released into the supernatants was about 90/10 for clone YP1, YP3 and Y strain, and 50/50 for clone YP2. After nine days, the ratio was 62/38 for clone YP1, 97/3 for clone YP3, 81/19 for Y strain and 50/50 for clone YP2. The susceptibility of tissue culture derived trypomastigotes (TCT) to lysis in the presence of chronic chagasic human sera and human complement was assessed using Complement Mediated Lysis reaction (CML). Trypomastigotes from clone YP2 were consistently less susceptible to CML (% lysis less than 20), than parasites from the other clones and Y strain. Parasites of clone YP3 had susceptibility to CML comparable to that of the Y strain (about 70%), while TCT of clone YP1 had intermediary susceptibility (40%).

CMRF-44 antibody-mediated depletion of activated human dendridic cells: A potential means for improving allograft survival

Background. Activated dendritic cells (DC) initiate immune responses by presenting antigen, including alloantigen from tissue grafts, to T lymphocytes. The potential to deplete or inactivate differentiated-activated DC during allogeneic transplantation represents a new approach to immunosuppression. Methods. The authors investigated the potential of the monoclonal antibody CMRF-44, which has specificity for a DC-associated differentiation-activation antigen, to induce complement-mediated lysis of activated human DC. Peripheral blood mononuclear cells (PBMC), or purified DC preparations, were cultured overnight to activate endogenous DC, resulting in the expression of CNW-44 antigen and CD83. These were then treated with CMRF-44 and complement. Depletion of activated DC was monitored by flow cytometry. Results. Eighty-nine percent of activated (CD83(+)) DC in cultured PBMC were depleted by treatment with CMRF-44 and autologous serum (AS) (complement source; mean percentage of CD83(+)-CD14(-)-CD19(-) cells=0.06%; cf 0.50% for heat-inactivated AS controls, P

Infectivity of amastigotes of Trypanosoma cruzi

The infectivity amastigotes of Trypanosoma cruzi, isolated from the supernatant of the J774G8 macrophage-like cell line infected with trypomastigotes to normal macrophages in vitro was tested. After a period of 1 h of T. cruzi-macrophage interaction about 2% of the mouse peritoneal macrophages had ingested amastigotes. In contrast 12% of the macrophages had ingested epimastigotes. Treatment of the amastigotes with trypsin did not interfere with their ingestion by macrophages. Once inside the macrophages the amastigotes divided and after some days transformed into trypomastigotes. When i.p. inoculated into mice the amastigotes were highly infective, inducing high levels of parasitaemia and tissue parasitism. As previously described for trypomastigotes, amastigotes were not lysed when incubated in the presence of fresh guinea-pig serum. Contrasting with what has been described for trypomastigotes, the resistance of amastigotes to complement-mediated lysis persisted after treatment with trypsin.

EVI antibodies in patients with Chagas' disease: relationship with anti-Trypanosoma cruzi immunoglobulins and effects of specific treatment

Antibodies against heart vascular structures and striated muscle cells interstitium (EVI antibodies) persist in Chagas' disease patients who had been cured by specific treatment as demonstrated by negative xenodiagnosis, conventional serology (CS) and complement mediated lysis (CoML). On the other hand, EVI antibodies are either present or absent in treated patients presenting positive CS but negative CoML. Since CoML detects antibodies associated to resistance, EVI antibodies are not likely to participate in the control of T. cruzi infections although they might be induced by cross-reacting antigens of heart cells and the parasite. They are neither necessarily related to antibodies responsible for CS. Absorption with T. cruzi and heart tissue confirms the suggestion that EVI antibodies are induced by a number of antigenic determinants, most from heart structures with a minor participation of T. cruzi antigens.

The utility of anti-trypomastigote lytic antibodies for determining cure of Trypanosoma cruzi infections in treated patients: an overview and perspectives

In previous work, we proposed alternative protocols for following patients with treated Chagas disease and these are reviewed herein. Evidence was provided to support the following: (i) functional anti-trypomastigote antibodies are indicative of ongoing chronic Trypanosoma cruzi infections; (ii) specific antibodies detected by conventional serology (CS) with epimastigote extracts, fixed trypomastigotes or other parasite antigens may circulate years after parasite elimination; (iii) functional antibodies are evidenced by complement-mediated lysis of freshly isolated trypomastigotes, a test which is 100% specific, highly sensitive, and the first to revert after T. cruzi elimination and (iv) the parasite target for the lytic antibodies is a glycoprotein of high molecular weight (gp160) anchored at the parasite surface. The complement regulatory protein has been cloned, sequenced and produced as a recombinant protein by other groups and is useful for identifying functional anti-T. cruzi antibodies in ELISA tests, thus dispensing with the need for live trypomastigotes to manage treated patients. If used instead of CS to define cures for Chagas patients, ELISA will avoid unnecessary delays in finding anti-T. cruzi drugs. Other highly sensitive techniques for parasite DNA detection, such as PCR, need to be standardized and included in future protocols for the management of patients with drug-treated Chagas disease.

Antitumour effects of single or combined monoclonal antibodies directed against membrane antigens expressed by human B cells leukaemia.

Background: The increasing availability of different monoclonal antibodies (mAbs) opens the way to more specific biologic therapy of cancer patients. However, despite the significant success of therapy in breast and ovarian carcinomas with anti-HER2 mAbs as well as in non-Hodkin B cell lymphomas with anti-CD20 mAbs, certain B cell malignancies such as B chronic lymphocytic leukaemia (B-CLL) respond poorly to anti-CD20 mAb, due to the low surface expression of this molecule. Thus, new mAbs adapted to each types of tumour will help to develop personalised mAb treatment. To this aim, we analyse the biological and therapeutic properties of three mAbs directed against the CD5, CD71 or HLA-DR molecules highly expressed on B-CLL cells. Results: The three mAbs, after purification and radiolabelling demonstrated high and specific binding capacity to various human leukaemia target cells. Further in vitro analysis showed that mAb anti-CD5 induced neither growth inhibition nor apoptosis, mAb anti-CD71 induced proliferation inhibition with no early sign of cell death and mAb anti-HLA-DR induced specific cell aggregation, but without evidence of apoptosis. All three mAbs induced various degrees of ADCC by NK cells, as well as phagocytosis by macrophages. Only the anti-HLA-DR mAb induced complement mediated lysis. Coincubation of different pairs of mAbs did not significantly modify the in vitro results. In contrast with these discrete and heterogeneous in vitro effects, in vivo the three mAbs demonstrated marked anti-tumour efficacy and prolongation of mice survival in two models of SCID mice, grafted either intraperitoneally or intravenously with the CD5 transfected JOK1-5.3 cells. This cell line was derived from a human hairy cell leukaemia, a type of malignancy known to have very similar biological properties as the B-CLL, whose cells constitutively express CD5. Interestingly, the combined injection of anti-CD5 with anti-HLA-DR or with anti-CD71 led to longer mouse survival, as compared to single mAb injection, up to complete inhibition of tumour growth in 100% mice treated with both anti-HLA-DR and anti-CD5. Conclusions: Altogether these data suggest that the combined use of two mAbs, such as anti-HLA-DR and anti-CD5, may significantly enhance their therapeutic potential.

Clearance-inducing antibodies are responsible for protection against the acute phase of Trypanosoma cruzi infection in mice

A study was conducted on mice infected with strains Y and CL of Trypanosoma cruzi. The ability of anti-Y and anti-CL sera to induce complement-mediated lysis, immune clearance and protection against the acute phase of the infection was studied using homologous anti-Y or anti-CL serum tested with the Y or CL strain, or heterologous anti-Y serum tested with the CL strain or anti-CL serum tested with the Y strain. Complement-mediated lysis was induced by both homologous and heterologous antisera but protection was afforded only by homologous antisera. Immune clearance was induced by homologous but not by heterologous antisera. Antisera with high clearance ability were able to confer protection whereas antisera with high lytic ability were not. These results show a high correlation between the antibody ability to induce clearance and to confer protection and suggest that clearance rather than lysis is responsible for protection against the acute phase of the infection. The mechanism of antibody protection against the acute phase of the infection is discussed.

Reduction in the level of Gal(alpha1,3)Gal in transgenic mice and pigs by the expression of an alpha(1,2)fucosyltransferase.

Hyperacute rejection of a porcine organ by higher primates is initiated by the binding of xenoreactive natural antibodies of the recipient to blood vessels in the graft leading to complement activation. The majority of these antibodies recognize the carbohydrate structure Gal(alphal,3)Gal (gal epitope) present on cells of pigs. It is possible that the removal or lowering of the number of gal epitopes on the graft endothelium could prevent hyperacute rejection. The Gal(alpha1,3) Gal structure is formed by the enzyme Galbeta1,4GlcNAc3-alpha-D-galactosyltransferase [alpha(1,3)GT; EC 2.4.1.51], which transfers a galactose molecule to terminal N-acetyllactosamine (N-lac) present on various glycoproteins and glycolipids. The N-lac structure might be utilized as an acceptor by other glycosyltransferases such as Galbeta1,4GlcNAc 6-alpha-D-sialyltransferase [alpha(2,6)ST], Galbeta1,4GlcNAc 3-alpha-D-Sialyltransferase [alpha(2,3)ST], or Galbeta 2-alpha-L-fucosyltransferase [alpha(1,2)FT; EC 2.4.1.691, etc. In this report we describe the competition between alpha(1,2)FT and alpha(1,3)GT in cells in culture and the generation of transgenic mice and transgenic pigs that express alpha(1,2)Fr leading to synthesis of Fucalpha,2Galbeta- (H antigen) and a concomitant decrease in the level of Gal(alpha1,3)Gal. As predicted, this resulted in reduced binding of xenoreactive natural antibodies to endothelial cells of transgenic mice and protection from complement mediated lysis.

Interaction of rheumatoid factor and Entamoeba histolytica

The amoebae's cytotoxicity test and the amoebae's lysis test were used to show possible interactions between rheumatoid factor (RF) and Entamoeba histolytica. Amoebae's cytotoxic activity (ACA) was inhibited by affinity chromatography purified antiamoebae rabbit IgG (RIgG). Enhanced inhibition could be demonstrated with RIgG plus RF. But the same marked inhibition of ACA could be seen when replacing RF by heat inactivated normal human serum as a control. About 50% amoebae's lysis occurred when amoebae were brought together with native normal human serum (NNHS) as a source of complement. Amoebae's lysis increased to 60% when incubated with NHS plus human antiamoebae antibodies. No further augmentation could be obtained by the addition of RF. Using RIgG instead of human antibodies the lysis rate did not increase. Incubation of amoebae, NNHS, RIgG and RF even reduced amoebae's lysis. RF neither has an effect on ACA nor on complement mediated AL in vitro.

The complement inhibitor low molecular weight dextran sulfate prevents TLR4-induced phenotypic and functional maturation of human dendritic cells.

Low molecular weight dextran sulfate (DXS) has been reported to inhibit the classical, alternative pathway as well as the mannan-binding lectin pathway of the complement system. Furthermore, it acts as an endothelial cell protectant inhibiting complement-mediated endothelial cell damage. Endothelial cells are covered with a layer of heparan sulfate (HS), which is rapidly released under conditions of inflammation and tissue injury. Soluble HS induces maturation of dendritic cells (DC) via TLR4. In this study, we show the inhibitory effect of DXS on human DC maturation. DXS significantly prevents phenotypic maturation of monocyte-derived DC and peripheral myeloid DC by inhibiting the up-regulation of CD40, CD80, CD83, CD86, ICAM-1, and HLA-DR and down-regulates DC-SIGN in response to HS or exogenous TLR ligands. DXS also inhibits the functional maturation of DC as demonstrated by reduced T cell proliferation, and strongly impairs secretion of the proinflammatory mediators IL-1beta, IL-6, IL-12p70, and TNF-alpha. Exposure to DXS leads to a reduced production of the complement component C1q and a decreased phagocytic activity, whereas C3 secretion is increased. Moreover, DXS was found to inhibit phosphorylation of IkappaB-alpha and activation of NF-kappaB. These findings suggest that DXS prevents TLR-induced maturation of human DC and may therefore be a useful reagent to impede the link between innate and adaptive immunity.

Hypoxia-inducible miR-210 regulates the susceptibility of tumor cells to lysis by cytotoxic T cells.

Hypoxia in the tumor microenvironment plays a central role in the evolution of immune escape mechanisms by tumor cells. In this study, we report the definition of miR-210 as a miRNA regulated by hypoxia in lung cancer and melanoma, documenting its involvement in blunting the susceptibility of tumor cells to lysis by antigen-specific cytotoxic T lymphocytes (CTL). miR-210 was induced in hypoxic zones of human tumor tissues. Its attenuation in hypoxic cells significantly restored susceptibility to autologous CTL-mediated lysis, independent of tumor cell recognition and CTL reactivity. A comprehensive approach using transcriptome analysis, argonaute protein immunoprecipitation, and luciferase reporter assay revealed that the genes PTPN1, HOXA1, and TP53I11 were miR-210 target genes regulated in hypoxic cells. In support of their primary importance in mediating the immunosuppressive effects of miR-210, coordinate silencing of PTPN1, HOXA1, and TP53I11 dramatically decreased tumor cell susceptibility to CTL-mediated lysis. Our findings show how miR-210 induction links hypoxia to immune escape from CTL-mediated lysis, by providing a mechanistic understanding of how this miRNA mediates immunosuppression in oxygen-deprived regions of tumors where cancer stem-like cells and metastatic cellular behaviors are known to evolve.

Ipilimumab-dependent cell-mediated cytotoxicity of regulatory T cells ex vivo by nonclassical monocytes in melanoma patients.

Enhancing immune responses with immune-modulatory monoclonal antibodies directed to inhibitory immune receptors is a promising modality in cancer therapy. Clinical efficacy has been demonstrated with antibodies blocking inhibitory immune checkpoints such as cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) or PD-1/PD-L1. Treatment with ipilimumab, a fully human CTLA-4-specific mAb, showed durable clinical efficacy in metastatic melanoma; its mechanism of action is, however, only partially understood. This is a study of 29 patients with advanced cutaneous melanoma treated with ipilimumab. We analyzed peripheral blood mononuclear cells (PBMCs) and matched melanoma metastases from 15 patients responding and 14 not responding to ipilimumab by multicolor flow cytometry, antibody-dependent cell-mediated cytotoxicity (ADCC) assay, and immunohistochemistry. PBMCs and matched tumor biopsies were collected 24 h before (i.e., baseline) and up to 4 wk after ipilimumab. Our findings show, to our knowledge for the first time, that ipilimumab can engage ex vivo FcγRIIIA (CD16)-expressing, nonclassical monocytes resulting in ADCC-mediated lysis of regulatory T cells (Tregs). In contrast, classical CD14(++)CD16(-) monocytes are unable to do so. Moreover, we show that patients responding to ipilimumab display significantly higher baseline peripheral frequencies of nonclassical monocytes compared with nonresponder patients. In the tumor microenvironment, responders have higher CD68(+)/CD163(+) macrophage ratios at baseline and show decreased Treg infiltration after treatment. Together, our results suggest that anti-CTLA-4 therapy may target Tregs in vivo. Larger translational studies are, however, warranted to substantiate this mechanism of action of ipilimumab in patients.