Schistosoma mansoni: Ultrastructural damages due to complement on schistosomula in vitro

The ultrastructural damage induced by complement in vitro on the schistosomula of Schistosoma mansoni was studied using transmission and scanning electron microscopy. The sequence of events which leads to the killing of the schistosomula is as follows: (a) the lytic activity starts at the anterior end of the schistosomula; (b) lesions progress simultaneously in two distinct directions: from the anterior to the posterior end, and from the outer membrane to the muscle layer; (c) “bubbles” appear around parasite which might correspond to increased membrane permeability; (d) the lytic activity of the late complement components occur in the syncytium; (e) the schistosomula lose their tegument completely, exposing the muscle layer. These findings and our previous work suggest that the activation of the alternate pathway and late complement components is sufficient, without antibodies or cells, to kill schistosomula in vitro.     

Eosinophil-mediated killing of schistosomula of Schistosoma mansoni: Oxidative requirement for enhancement by eosinophil colony stimulating factor (CSF-α) and supernatants with eosinophil cytotoxicity enhancing activity (E-CEA)

Factors which enhance eosinophil-mediated killing of antibody-coated schistosomula of Schistosoma mansoni include semipurified eosinophil colony stimulating factor (CSF-α) and eosinophil cytotoxicity enhancing activity (E-CEA) present in supernatants from cultured mononuclear cells. We have examined the mechanism of enhancement. Both actions require oxygen in order to enhance killing and do not enhance killing under anaerobic conditions (P ? 0.005). E-CEA had no detectable effect upon oxidative metabolism. In contrast to CSF-α which, in our previous studies, increased Superoxide anion productions and quantitative leukocyte iodination, E-CEA had no detectable effect upon oxidative metabolism. In order to test whether active oxygen products might mediate enhancement of killing, the effects of the addition of Superoxide dismutase and catalase were tested. Neither enzyme showed inhibition of CSF-α or E-CEA enhancement of eosinophil-mediated killing. The effects of CSF-α and E-CEA were not additive. These studies suggest that both CSF-α and E-CEA exert enhancement of killing by means of an as yet unidentified oxygen requiring process.     

Schistosoma mansoni: Complement and antibody damage,mediated by human eosinophils and neutrophils,in killing schistosomula in vitro

The time and course of damage to Schistosoma mansoni schistosomula mediated by human eosinophils and neutrophils and by antibody (A) and/or complement (C) was studied. The rate of schistosomula death was significantly higher in the complement containing systems (i.e., “A + C” or “C alone”) when compared to A alone. In general, at all the time points studied, the percentage of killing in the three systems was A + C > C alone > A alone irrespective of whether the effector cells were neutrophils or eosinophils. Preferential killing of schistosomula by eosinophils, as compared to neutrophils, was observed with C alone and A + C, but only when eosinophils and neutrophils from the same donor were compared. In contrast, eosinophils and neutrophils appeared to be equally effective in killing antibody-coated schistosomula.A comparison was made of the susceptibility to killing of schistosomula prepared mechanically or by skin penetration. There was no appreciable difference in terms of susceptibility to killing by the various combinations of eosinophils, neutrophils, antibody, and complement between these two forms of schistosomula.The preferential killing of complement-coated, as compared to antibody-coated schistosomula by eosinophils appears to be relatively rapid, an observation which may be of significance both in natural and acquired immunity to migrating larval helminths.     

Cytotoxicity of white blood cells activated by granulocyte-colony-stimulating factor,granulocyte/macrophage-colony-stimulating factor and macrophage-colony-stimulating factor against tumor cells in the presence of various monoclonal antibodies

Unconjugated monoclonal antibodies (mAb) kill tumor cells in vivo by activating immune functions. One of these is ADCC (antibody-dependent cellular cytotoxicity). The efficacy of mAbs might be augmented if the cytotoxic capacity of the effector cells could be increased. In this study the augmenting effect of granulocyte-colony-stimulating factor (G-CSF), granulocyte/macrophage(GM)-CSF and macrophage(M)-CSF was analyzed. Effector cells [peripheral blood mononuclear cells (PBMC) or granulocytes] were activated for 4–6 h by the respective CSF and assayed in an 18-h Cr⁵¹-release assay. Human colorectal, lymphoma, glioma and melanoma cell lines were target cells. Mouse mAbs of different isotypes, as well as chimeric and humanized mAbs, were used. mAbs having the human Fc part of the IgG molecule were the most effective. The killing capacity of PBMC as well as of granulocytes was statistically significantly enhanced when mAbs were added. M-CSF and GM-CSF were the best CSF for augmenting the lytic capacity of PBMC in ADCC. G-CSF had no significant effect on PBMC. Spontaneous cytolysis of PBMC was significantly augmented only by M-CSF. Granulocytes were, in general, significantly less effective than PBMC but may be equally effective killer cells together with mouse or human mAbs of the IgG1 isotype, particularly against melanoma cells. Granulocytes may also be significantly stimulated to increased lytic capacity when activated with G-CSF or GM-CSF. On the basis of the present evaluation, clinical trials in tumor patients are warranted, combining mAbs with GM-CSF or M-CSF. Preference might be given to GM-CSF as this cytokine activates both PBMC and granulocytes.     

Liquid and cryopreservation effects on in vitro function of dog granulocyte concentrates prepared for transfusion

The effects of liquid and Cryopreservation on in vitro function of dog granulocyte concentrates prepared by continuous flow centrifugation leukapheresis and counterflow centrifugation elutriation are presented. These homogeneous granulocyte concentrates (97 ± 2% granulocytes, 99.4 ± 0.3% viable) were cryopreserved in 5% DMSO and 5% HES dissolved in 2 g% BSA, 20% autologous citrated plasma in a modified a minimal essential medium. The granulocyte recovery was 87.6 ± 2.4% relative to the number of intact and viable granulocytes in the washed suspension of cells. In vitro functions of chemotaxis, phagocytosis, bactericidal activity, and selected enzymes were not affected by 12–24 hr storage at 4–6 °C. Frozen, thawed, and washed granulocytes showed a significant decrease (P < 0.01) in chemotactic recognition and response but not chemokinetic response, although it was depressed. Phagocytosis of latex beads and associated burst of O₂ consumption also decreased significantly (P < 0.05) to approximately 50% of the original prefreezing value. However, the killing of live Escherichia coli was not depressed to the extent expected and suggested by loss of O₂ consumption and selected enzyme activity. The cryopreservation of viable homogeneous granulocyte concentrates in sufficient quantity for transfusion in the neutropenic and/or septicemic dog model is demonstrated in these results.     

Immunomagnetic bead separation of mononuclear cells from contaminating granulocytes in cryopreserved blood samples

Density gradient centrifugation usually allows efficient separation of mononuclear cells from granulocytes using fresh human blood samples. However, we have found that with cryopreserved blood samples, density gradient centrifugation fails to separate granulocytes from mononuclear cells and have explored using immunomagnetic anti-CD15 microbeads as an alternate method to separate these cell populations. Using cryopreserved blood samples from 10 healthy donors we have shown that granulocytes express a significantly higher level of CD15 antigen than monocytes and lymphocytes, which allows for their efficient separation from mononuclear cells using anti-CD15 microbeads. This procedure is critical for purification of individual cell populations from cryopreserved leukocyte samples and could also potentially be applied to avoid granulocyte contamination of mononuclear cells isolated from stored blood and from patients with sepsis or thermal injury.     

Antibody-dependent murine macrophage-mediated damage to Schistosoma mansoni schistosomula in vitro

Antibody-dependent cell-mediated cytotoxicity (ADCC) against schistosomula of the human parasite Schistosoma mansoni was demonstrated using antisera from mice plus peritoneal exudate cells (PEC). PEC were divided into plastic-adherent (96% macrophages, 4% lymphocytes) and nonadherent (92% lymphocytes, 8% macrophages) cell populations. Four criteria of ADCC were used, including minimal and maximal cell attachment, and death of and uptake of trypan blue by schistosomula. Using cells from normal mice and antisera from schistosome-infected mice, macrophages adhered to, damaged the tegument and underlying structures of, and killed schistosomula when observed following 18 hr incubation. In homologous systems, the results were similar when outbred CD-1 and inbred BALB/c mice were compared, except that potency of antisera from the latter mice decreased after 6–7 weeks postinfection, whereas the opposite was true for the former strain of mice. Nonadherent cells also exhibited ADCC against schistosomula, but the potency was considerably lower than that of adherent cells. These complement-independent ADCC reactions were stage-specific for the schistosomulum in that no reactions occurred with adult worms.     

A deficiency in the B cell response of C57BL/6 mice correlates with loss of macrophage-mediated killing of Leishmania amazonensis

Infection of C3HeB/FeJ and C57BL/6 mice with Leishmania major stimulates a healing cell-mediated immune response, while Leishmania amazonensis infection leads to chronic disease. Here we show C3HeB/FeJ mice co-infected with both species of Leishmania heal, while co-infected C57BL/6 mice do not. Using an in vitro killing assay we determined B cells from infected C57BL/6 mice are ineffective in promoting parasite killing compared with B cells from infected C3HeB/FeJ mice. Furthermore, infected C57BL/6 mice produce less antigen-specific antibodies compared with infected C3HeB/FeJ mice. These findings suggest B cells play a required role in the cell-mediated immune response against L. amazonensis.     

Neutrophil migration in canines: In vivo comparison of granulocyte function following 24-hour storage at 6 or 20 °C of leukocyte concentrates or of granulocytes purified by counterflow centrifugation-elutriation

The use of granulocyte-rich concentrates from leukapheresis purified by counterflow centrifugation—elutriation to obtain pure granulocytes for transfusion studies in cyclo-phosphamide-induced neutropenic animal models is reported. Our data for granulocyterich leukapheresis concentrates indicate that room temperature (20 °C) appears to be preferred to 6 °C for short-term granulocyte storage. The data also indicate that although the granulocytes isolated by counterflow centrifugation—elutration may retain in vitro functions of chemotaxis, phagocytosis, and bactericidal activity, the in vivo function of migration into skin chambers for isolated granulocytes is seriously impaired after storage for 18 to 24 hr at both 6 and 20 °C. This loss of in vivo function of stored granulocytes occurs in isolated granulocytes obtained by both counterflow centrifugation-elutriation and dextran sedimentation, and it is not observed in the leukocyte concentrates held at 20 °C. The results of these studies are fourfold. First, freshly isolated granulocytes display no apparent loss of either in vivo or in vitro function. Second, granulocytes isolated by counterflow centrifugation-elutriation or dextran sedimentation and stored at 6 or 20 °C are severely impaired in terms of their in vivo chemotactic function but display no loss of in vitro efficacy. Third, 20 °C storage of granulocyte-rich leukapheresis concentrates for 18 to 24 hr is superior to 6 °C storage. Fourth, in vitro analysis may be limited in its ability to indicate in vivo function as a measure of success in granulocyte preservation studies.     

Schistosoma mansoni: Cryopreservation of schistosomula by two-step addition of ethanediol and rapid cooling

A simple, inexpensive, and highly effective technique for the Cryopreservation of schistosomula of Schistosoma mansoni is outlined by experiments designed to clarify the role of each of the steps involved. The technique consists of incubating schistosomula in 10% ($\text{v}\text{v}$) ethanediol for 10 min at 37 C followed by 5 min at 0 C and for a further 10 min in 35% ($\text{v}\text{v}$) ethanediol at 0 C. The schistosomular suspension is then aliquotted in 20-μl drops onto 40 × 5.5-mm glass slivers prepared from standard microscope coverslips, each drop being spread out to cover an area of approximately 15 × 4 mm. These glass slivers are then dropped directly into liquid nitrogen giving a cooling rate of approximately 5000 C min^?1. Survival is further improved if the schistosomula are at least 90 min old before Cryopreservation and if the frozen organisms are thawed in culture medium prewarmed to +42 C. Levels of survival obtained with this technique are consistently high: 44 to 60% as assessed by motility. From 400 ± 11 cryopreserved schistosomula injected intramuscularly into eight mice, a mean of 54.5 ± 16.3 adult worms were recovered representing an infection level of 13.7%, and which in turn represents 47.4% of the unfrozen control level.     

The effects of immune rhesus monkey serum on schistosomula of Schistosoma mansoni during cultivation in vitro

Clegg J. A. and Smithers S. R., 1972. The effect of immune rhesus monkey serum on schistosomula of Schistosoma mansoni during cultivation in vitro. International journal for Parasitology2: 79–98. The sera of rhesus monkeys hyperimmunized by 2–4 exposures to S. mansoni cercariae contain an antibody lethal to schistosomula cultivated in vitro. The antibody (IgG) is dependent on labile factors in fresh monkey serum. It can be absorbed by adult worms cultivated in vitro and it is not the antibody responsible for CHR or COP reactions. A titre of lethal antibody sufficient to kill all schistosomula in vitro is maintained for 2–3 months following challenge: it then falls to a moderate level which may be retained for several years. After inactivation, hyperimmune serum inhibits the growth of cultured schistosomula but does not kill them. Following a small primary infection rhesus serum develops a marked growth-inhibiting property and a low titre of lethal antibody at about 4 months, i.e. the time when resistance to reinfection can first be reliably demonstrated.     

Unaltered hematocrit values and differential hemocyte counts in acanthocephalan-infected Armadillidium vulgare

Three cell types were present in Armadillidium vulgare hemolymph. Hyaline cells, 8.2 to 12.0 μm in size with a few fine granules in the cytoplasm, comprised 42% of the cells. Twelve percent of the cells were nonexplosive granulocytes, 8.8 to 15.0 μm, with many cytoplasmic granules of medium size. A third cell type, an explosive 7.2- to 12.0-μm granulocyte with coarse cytoplasmic granules, comprised 46% of the cells. Within 48 hr after adult specimens of A. vulgare were fed eggs of the acanthocephalan, Plagiorhynchus cylindraceus, isopod hemocytes aggregated in the intestinal epithelium surrounding penetrating acanthors. Encapsulation and death of the parasite routinely followed. No significant difference in hematocrit values or in differential hemocyte counts occurred between infected and uninfected control isopods.     

Optimized radiolabelling of pig granulocytes with indium-111 tropolonate

Standard labelling methods using ¹¹¹In-tropolonate in plasma resulted in lower labelling efficiency (LE) with pig granulocytes than with human cells. Addition of acid-citrate-dextrose (ACD) to cell-free plasma in a ratio of 1:10 tripled the LE to a value which was not significantly different from that obtained in saline. Under optimized conditions, LE was 67±2% (mean±SE) with pig granulocytes, which remained lower than that obtained with human cells but was adequate for nuclear imaging studies of granulocyte deposition in the pig.     

Antibody-dependent cytotoxicity of human and mouse mononuclear cells against Trypanosoma cruzi epimastigotes

Human peripheral mononuclear cells were cytotoxic to antibody-sensitized Trypanosoma cruzi epimastigotes. The cytotoxic effect depended on the concentration of effector cells and antiserum, and was progressive until 17 hr of incubation at 28 °C. After 3 hr of incubation the highest specific activity was achieved at a 50:1 effector to target cell ratio. A nonspecific cytotoxic effect in the absence of antiserum was observed at a 100:1 parasite to cell ratio or after 17 hr of incubation. When the human mononuclear cell population was depleted of adherent cells by Sephadex G-10 filtration or adsorption to glass, the cytotoxic effect was greatly reduced. Similar results were obtained using mouse spleen cells, indicating that only the adherent cells were cytotoxic to sensitized T. cruzi in both systems. When human mononuclear cells were incubated with amobarbital, cyanide, azide, or aminotriazole, an inhibition of cytotoxicity against sensitized T. cruzi was observed, suggesting that oxygen reduction products and myeloperoxidase were involved in the destruction of sensitized T. cruzi epimastigotes by normal human mononuclear cells.     

In vitro immune mechanisms associated with clearance of microfilariae of Dirofilaria immitis

An in vitro system has been developed to elucidate potential immune mechanisms associated with clearance of microfilariae (Mf) from the bloodstream in canine Dirofilaria immitis infection. Granulocytes as well as mononuclear cells adhere to Mf of Dirofilaria immitis in the presence of immune serum. Only granulocytes, however, were capable of killing Mf, whereas PBMC attach to but do not effectively kill Mf. In the presence of granulocytes 1% +/- 1, 10% +/- 2, and 12% +/- 3 of Mf were killed by heated normal (NDS), patent (PS), and occult serum (OS), respectively, after an 18-hr incubation. With the addition of fresh NDS there was an increase in killing to 5% +/- 1 (p less than 0.025) with heat-inactivated NDS, to 12% +/- 3 in the presence of PS and to 77% +/- 12 (p less than 0.005) in the presence of OS. On further purification of granulocyte cell populations with metrizamide gradients, neutrophils were found to be the predominant effector cells with 73% +/- 18 killing with neutrophils and 18% +/- 6 with eosinophils (p less than 0.0005). Only with neutrophils was a significant increase in killing of Mf observed when fresh NDS was added to delta OS. Fractionation of OS by gel filtration suggested that IgM was the opsonizing antibody in the occult serum. In addition, immunofluorescent studies showed only IgM bound to the surface on Mf on incubation in OS. The involvement of complement in the fresh serum enhancement of killing was supported by the finding, by immunofluorescence, of surface C3 on Mf after incubation in fresh OS.     

Bactericidal activity of coelomic fluid from the sea urchin Echinus Esculentus

Coelomic fluid from the common British sea urchin Echinus esculentus was shown to kill in vitro a black-colonied, agar-digesting marine Pseudomonas sp. (strain No. 111). The bactericidal reaction, which was monitored by viability counting on marine agar plates, took 48 hr to reach full expression. An incubation temperature of 4°C gave the most complete and consistent killing. Clotting of the coelomocytes did not influence bactericidal activity. The vast majority of nearly 200 specimens of E. esculentus from the Clyde Sea area of Scotland yielded coelomic fluid with bactericidal activity, and there was no apparent seasonal variation. Activity was localized in the coelomocytes and could be obtained in cell-free extracts by ultrasonic disruption of these cells. The bactericidal activity in these extracts was lost during dialysis but was only partially destroyed by heating for 30 min at 56° or 100°C.     

Failure of anti-Fasciola antisera to induce antibody-dependent, eosinophil-mediated killing of Schistosoma mansoni schistosomula

Sera from rabbits or humans infected with Fasciola hepatica were tested for their ability to kill Schistosoma mansoni schistosomula in an antibody-dependent, eosinophil-mediated in vitro assay. In addition, anti-F. hepatica antisera raised in rabbits or calves, including one to a Fasciola/Schistosoma cross-reactive, cross-protective defined immunity antigen, were also tested for their killing ability. None of these antisera induced damage to S. mansoni schistosomula in vitro, even when enhanced with mononuclear cell supernatants containing eosinophil-activating factor. Serum from humans with S. mansoni did induce schistosomulum killing in vitro when tested under these same conditions. These results suggest that the mechanism of immunity to schistosomes induced by Fasciola antigens at the level of the schistosomula is mediated by factors other than eosinophils.     

A technique for the separation and cryopreservation of myeloid stem cells from human bone marrow.

We have developed a technique for the cryopreservation of large volumes of human bone marrow, which reduces cell losses due to clumping and release of lysosomal enzymes from mature granulocytes. Mononuclear cells were separated from whole bone marrow by a large-scale Ficoll-Hypaque procedure. The agar colony assay for myeloid stem cells (CFU-C) was used to assess each step of the isolation and cryopreservation procedure. Conditions of varied cell and cryoprotectant concentrations and freezing and thawing rates were compared to obtain optimal recovery of mononuclear cells and CFU-C. This technique has been used to store bone marrow from 45 patients with hematologic and non-hematologic neoplasms. Up to 750 ml of marrow was obtained from each patient and separated by step-gradient centrifugation, and the cell fraction containing myeloid stem cells was cryopreserved. The mean recoveries following separation, cryopreservation, and thawing for 18 marrow storages from patients with hematological neoplasms were 8.8 ± 2.9% for mononuclear cells and 47.8 ± 20.8% for CFU-C. In comparison, values for 27 marrows from patients with non-hematological neoplasms were 14.5 ± 5.5% for cells and 57.7 ± 13.7% for CFU-C.     

Multidimensional flow-cytometric analysis of dendritic cells in peripheral blood of normal donors and cancer patients

 We studied the potential of multidimensional flow cytometry to evaluate the frequency and maturation/activation status of dendritic cells in minimally manipulated peripheral blood mononuclear cell preparations (i.e., only separated on Ficoll-Hypaque) of normal donors and cancer patients. A rare subset of HLA-DR⁺ leukocytes (less than 1% mononuclear cells) was detected in blood of normal donors that displayed all the features of dendritic cells: these cells had high forward-light-scatter characteristics and coexpressed CD4, CD86 and CD54 surface antigens, but lacked the lineage-associated surface markers of T cells, B cells, monocytes, granulocytes or NK i.e. they were CD3^–, CD19^–, CD20^–, CD14^–, CD11b^–, CD16^–, CD56^–). These physical and phenotypic properties were virtually identical to those of immunomagnetically sorted leukocytes characterized as dendritic-cells on the basis of morphology, phenotype and high stimulatory activity in allogeneic mixed-lymphocyte cultures. Using this flow-cytometric approach we observed that the frequency of dendritic cell-like cells in peripheral blood mononuclear cell specimens of cancer patients receiving chemotherapy alone or those recovering from stem cell transplantation was significantly lower than that of normal individuals (mean ± SE: 0.36 ± 0.05%, 0.14 ± 0.06%, and 0.75 ± 0.04% respectively). Multidimensional flow-cytometric analysis of dendritic cells might represent an important new tool for assessing immunocompetence, and for monitoring the effects of therapeutic regimens on the immune system. Received: 20 June 1997 / Accepted: 14 August 1997     

The presence of antibody in mice chronically infected with Schistosoma mansoni which blocks in vitro killing of schistosomula

We have previously reported that IgM monoclonal antibodies (mAb) that recognize surface carbohydrate determinants shared between schistosomula, cercariae, and miracidia block antibody/complement dependent killing of schistosomula in vitro. Binding assays that make use of one of the IgM mAb labeled with 125I demonstrated that serum from chronically infected mice (CMS) contained high levels of competing antibody, whereas serum from mice vaccinated with irradiated cercariae (VMS) contained little antibody of this specificity. Absorption of CMS with cercariae that removed antibodies to schistosomulum surface carbohydrate determinants increased its ability to kill schistosomula in vitro; absorption of VMS with cercariae failed to alter the lethal activity of the serum. Furthermore, fractionation of CMS by protein A Sepharose chromatography demonstrated that the IgG fraction had an increased lethal activity compared with unfractionated serum; this result was not seen with VMS. Finally, the IgM fraction of CMS was shown to block in vitro killing of the IgG fractions of both CMS and VMS. These data suggest that the blocking activities observed with the IgM mAb are contained within the serum of chronically infected mice but not in the serum of mice vaccinated with irradiated cercariae.