In vitro killing of microfilariae of Brugia pahangi and Brugia malayi by eosinophil granule proteins

Eosinophil infiltration and degranulation around the tissue-invasive stages of several species of helminths have been observed. Release of eosinophil granule contents upon the worms is supported by localization of two of the major granule proteins, major basic protein (MBP) and eosinophil peroxidase (EPO), on and around species of trematodes, nematodes, and cestodes. In the case of filarial worms, MBP is deposited on degenerating microfilariae (mf) of Onchocerca volvulus. Here, we performed in vitro assays of the toxicity of four purified eosinophil granule proteins, namely, MBP, EPO, eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN), for the mf of Brugia pahangi and Brugia malayi. MBP, ECP, and EDN killed these worms in a dose-related manner although relatively high concentrations of EDN were necessary. EPO, in the presence of a H2O2-generating system and a halide, was the most potent toxin on a molar basis; here, the most potent halide was I- followed by Br- and Cl-. Surprisingly, EPO in the absence of H2O2 killed mf at concentrations comparable to those required for MBP and ECP. The toxicity of EPO + H2O2 + halide was inhibited by heparin, catalase, or 1% BSA, whereas the toxicity of EPO alone was inhibited only by heparin. Heparin also inhibited killing by both MBP and ECP. Despite the homology of ECP with certain RNases, placental RNasin, an RNase inhibitor, was unable to inhibit ECP-mediated toxicity. These results indicate that all of the eosinophil granule proteins are toxic to mf and they support the hypothesis that eosinophil degranulation causes death of mf in vivo.     

Induction of antigen-specific CTL by recombinant HIV trans-activating fusion protein-pulsed human monocyte-derived dendritic cells

Several systems have been tested for introduction of Ags into human dendritic cells (DC). Most of them to date, however, are complex and possess limited efficiency. Recent advances in HIV trans-activating (TAT) fusion protein technology permit extremely high transduction efficiencies for a majority of mammalian cell types. Here we report our attempts to develop a simple, but highly efficient, protocol for loading of antigenic protein into DC using TAT fusion technology. A TAT-minigene fusion protein was generated, encoding both the HLA-A2-restricted influenza matrix protein-derived epitope (GILVFTFTL, Flu-M1) and a melanoma Ag gp100-derived modified epitope (YLEPGPVTV, G9(280)-9V). In addition, both a TAT-Her2/neu extracellular domain (ECD) fusion protein and a TAT-green fluorescence protein fusion protein were generated. Over 95% of DC stained positively for TAT-green fluorescence protein within 20 min of coculture. DC treated with TAT-minigene were efficiently recognized by both Flu-M1 and G9(280)-9V-specific T cells in cytotoxicity assays and IFN-gamma ELISPOT assays. In contrast, DC pulsed with minigene fusion protein lacking TAT were either poorly recognized or not recognized by the T cells. DC pulsed with TAT-minigene also efficiently induced Flu-M1-specific T cells from naive lymphocytes. Similarly, DC treated with TAT-Her2/neu ECD stimulated patient-derived lymphocytes that specifically recognized Her2/neu(+) ovarian and breast cancer cell lines. The CTL induced by TAT-Her2/neu ECD-pulsed DC specifically recognized the Her2/neu ECD-derived immunogenic peptide E75 (KIFGSLAFL). Our data suggest that TAT fusion proteins efficiently transduce DC and induce Ag-specific T cells. This could prove to be a useful method for treatment of infectious diseases and cancer.     

Mucin-induced apoptosis of monocyte-derived dendritic cells during maturation

Many tumors arising from epithelial tissues produce mucins, which readily come into contact with infiltrating cells in cancer tissues. MUC2 mucins were purified from the conditioned medium of a colorectal cancer cell line, LS180 cells. It is known that in cancer patients, the number of dendritic cells (DCs) is reduced and their function is impaired. Mature DCs were generated from human peripheral blood monocytes through successive treatments with GM-CSF and IL-4, and then with proinflammatory mediators. When monocytes were cultured in the presence of MUC2 mucins in addition to GM-CSF and IL-4 at an early stage of development, mature DCs expressing CD83 decreased and apoptotic cells increased in a dose-dependent manner. During the development of DCs, sialic acid-binding Ig-like lectin (Siglec)-3 was constantly expressed. We prepared recombinant soluble Siglec-3 corresponding to the ectodomain of Siglec-3 and confirmed the binding of soluble Siglec-3 to the MUC2 mucins, probably through alpha2,6-sialic acid-containing O-glycans including a sialyl Tn antigen, which is known to bind to Siglec-3. Apoptosis was partially inhibited by anti-Siglec-3 mAb or recombinant soluble Siglec-3. These results suggest that apoptosis was partially induced through the ligation of the MUC2 mucins with Siglec-3.     

Induction of protection against Brugia malayi infection in jirds by microfilarial antigens

The development of immunologic methods to reduce transmission of human lymphatic filariasis depends on measures that will enhance the host's ability to eliminate infective larvae, adult worms, or blood-borne microfilariae (mf). The present study was designed to assess the capacity of a crude extract of Brugia malayi mf to decrease the level of microfilaremia and adult worm burden in jirds inoculated with infective larvae, and to identify the filarial antigens that elicit antibody responses in these animals. Thirty weeks after subcutaneous inoculation with 75 infective larvae, 100% of control jirds were patent (i.e., had microfilaremia) compared with 60% of the group immunized with 10 micrograms of crude microfilarial extract (p less than 0.05). In addition, microfilaremia was lower in patent immunized animals compared with controls (p less than 0.05). The mean total number of adult female B. malayi per jird recovered at necropsy in control animals was 16.0 vs 7.0 in immunized jirds (p less than 0.05). Serum of immunized jirds contained anti-mf antibodies with an end titer of 1:8000, a value similar to that of animals with chronic B. malayi infection. Microfilarial antigens of Mr approximately 150,000, 75,000, 42,000, and 25,000 were identified in immunoblotting studies by reactivity with antibodies in sera of immunized jirds. Antibodies induced by immunization with microfilarial extract were not specific for this stage of the parasite life cycle, as jird anti-mf antibodies reacted with a Mr approximately 150,000 and several Mr 50,000 to 110,000 antigens derived from immature and mature adult parasites of both sexes. These data indicate that immunization of jirds with a water soluble microfilarial extract enhances the host's ability to eliminate adult worms and blood-borne mf. The filarial antigens that induce antibodies in immunized jirds have been identified.     

Gene expression analysis in human monocytes, monocyte-derived dendritic cells, and alpha-galactosylceramide-pulsed monocyte-derived dendritic cells.

In vitro proliferation and functional activation of V alpha 24NKT cells following stimulation with alpha-galactosylceramide (alpha-GalCer)-pulsed dendritic cells (DCs) have been observed. Because little is known about the molecular events on DCs following interaction with alpha-GalCer, we performed gene expression profiling of 2400 genes in monocytes and monocyte-derived immature DCs pulsed with alpha-GalCer (alpha-GalCer-imDCs). Overall, the expression levels of 48 genes were up-regulated and 28 were down-regulated in alpha-GalCer-imDCs. Semiquantitative RT-PCR analysis on monocytes, imDCs, alpha-GalCer-imDCs, and mature DCs confirmed the up- and down-regulation of the mRNA expression levels of 28 selected genes. Notably, we identified the specific up-regulation of mRNA expression levels of ribonuclease A and collapsin response mediator protein upon the stimulation of imDC with alpha-GalCer, suggesting a novel immunomodulating effect of alpha-GalCer on imDCs. In this study, we used imDCs prepared by culturing of monocytes with GM-CSF and IL-4 for 5 days and mDCs prepared by further culturing of imDCs with TNF alpha for two extra days.     

Brugia malayi microfilariae induce cell death in human dendritic cells,inhibit their ability to make IL-12 and IL-10, and reduce their capacity to activate CD4+ T cells

Parasite Ag-specific T cell unresponsiveness and diminished IFN-gamma production are immunologic hallmarks of patent infection with lymph-dwelling filarial nematodes. Although this diminished responsiveness is directed primarily against the intravascular microfilarial (MF) parasite stage and mediated in part by reduced APC function, the mechanisms involved are not fully understood. In this report, we demonstrate that human dendritic cells (DC) exposed to live MF up-regulate both the cell surface and gene expression of CD54 (ICAM-1). Moreover, live MF result in a 3-fold increase in DC death compared with MF-unexposed DC, primarily due to apoptosis. Notably, microarray and real-time RT-PCR data indicate that live MF concurrently up-regulate mRNA expression of proinflammatory molecules such as IL-8, RANTES, IL-1alpha, TNF-alpha, and IL-beta in DC, the presence of which is also detected at the protein level, while inhibiting the production of IL-12 (p40 and p70) and IL-10. Soluble excretory-secretory products from live MF diminished IL-12 and IL-10 production and induced DC death, although to a lesser degree. Moreover, exposure of DC to live MF resulted in a decrease in the ability of DC to promote CD4(+) T cell production of IFN-gamma and IL-5. Our findings clearly suggest that the interaction between live MF and DC is complex but contributes to the hyporesponsiveness and parasite persistence associated with the MF(+) state in the infected human. These data further suggest that MF induce an orchestrated response in APC that leads to a diminished capacity to function appropriately, which in turn has significant consequences for CD4(+) T cells.     

Nucleoporation of dendritic cells: efficient gene transfer by electroporation into human monocyte-derived dendritic cells

Four new members of the ERF (ethylene-response factor) family of plant-specific DNA-binding (GCC box) factors were isolated from tomato fruit (LeERF1–4). Phylogenetic analysis indicated that LeERF2 belongs to a new ERF class, characterized by a conserved N-terminal signature sequence. Expression patterns and cis/trans binding affinities differed between the LeERFs. Combining experimental data and modeled three-dimensional analysis, it was shown that binding affinity of the LeERFs was affected by both the variation of nucleotides surrounding the DNA cis-element sequence and the nature of critical amino acid residues within the ERF domain.     

Brugia malayi: Effects of nitazoxanide and tizoxanide on adult worms and microfilariae of filarial nematodes

There is an urgent need for safe and effective antifilarials. Prior studies have shown that the nitazoxanide (NTZ) exhibits broad activity against anaerobic bacteria, protozoa, and certain intestinal helminths. We examined the effects of NTZ and tizoxanide (TZ) on Brugia malayi nematodes in vitro and in vivo. In vitro, NTZ and TZ reduced worm motility and viability in a dose-dependent manner. Worm viability was reduced by 50% with both compounds at 2.5 and 20 μg/ml killed adult worms. NTZ or TZ (5 μg/ml) significantly reduced microfilaria release. These compounds blocked worm’s embryogenesis, and decreased microfilarial motility and viability. Treated worms had damaged cuticles and abnormal mitochondria. Wolbachia were not cleared by NTZ or TZ treatment. Neither NTZ nor TZ cleared adult worms or microfilariae in infected gerbils. These results show that NTZ and TZ have potent effects on B. malayi nematodes in vitro. However, they were not effective in vivo.     

Tumors promote altered maturation and early apoptosis of monocyte-derived dendritic cells

Tumors produce a number of immunosuppressive factors that block the maturation of CD34+ stem cells into dendritic cells (DC). We hypothesized that tumors might also interfere with the maturation and/or function of human monocyte-derived DC. In contrast to stem cells, we found that CD14+ cells responded to tumor culture supernatant (TSN) by increasing expression of APC surface markers, up-regulating nuclear translocation of RelB, and developing allostimulatory activity. Although displaying these characteristics of mature DC, TSN-exposed DC lacked the capacity to produce IL-12, did not acquire full allostimulatory activity, and rapidly underwent apoptosis. The effects of TSN appeared to be specific for maturing DC, and were not reversed by Abs against known DC regulatory factors including IL-10, vascular endothelial growth factor, TGF-beta, or PGE2. Supernatants collected from nonmalignant cell sources had no effect on DC maturation. The altered maturation and early apoptosis of monocyte-derived DC may represent another mechanism by which tumors evade immune detection.     

Brugia malayi: stage-specific expression of carbohydrates containing N-acetyl-D-glucosamine on the sheathed surfaces of microfilariae

Microfilariae, infective larvae, and adult worms of Brugia malayi were incubated with a panel of seven lectins in order to study the expression of surface carbohydrates. Infective larvae and adult worms did not bind any of the lectins utilized. Microfilariae, on the other hand, bound wheat germ agglutinin. The binding of this lectin was saturable and specific, and attributed to the presence of N-acetyl-D-glucosamine. In addition, microfilariae derived in vitro bound concanavalin A, indicating the presence of glucose and/or mannose on this stage of the parasite. The fact that similar concanavalin A binding was not seen on microfilariae recovered directly from the infected host implies that there is masking or loss of parasite surface antigens as microfilariae mature in vivo.     

Brugia malayi: intravenous injection of microfilariae in ferrets as an experimental method for occult filariasis

Microfilaremia, immune responses, and pathology were compared in ferrets infected with 100 third-stage larvae of Brugia malayi (subperiodic strain) or injected intravenously with 10(6) microfilariae. Ferrets (Mustela putorius furo) inoculated with third-stage larvae typically became patent during the third month after infection, with a mean patency of 123 +/- 25 (SE) days. Ferrets injected intravenously with microfilariae exhibited a relatively constant microfilaremia for 3-4 weeks and usually cleared microfilariae before the fourth month. Ferrets that cleared microfilariae after intravenous injection of microfilariae or after infection with third-stage larvae failed to become patent or became amicrofilaremic within 3 weeks after a challenge intravenous injection of 10(6) microfilariae. Clearance of circulating microfilariae was associated with eosinophilia and serum antibody specific for the microfilarial sheath in ferrets injected with microfilariae and in most ferrets infected with third-stage larvae. Ferrets infected with third-stage larvae and necropsied after clearance of microfilariae had tissue inflammatory reactions to microfilariae characteristic of occult filariasis (tropical eosinophilia) in man; these ferrets exhibited immediate cutaneous hypersensitivity and circulating reaginic antibody to antigens of microfilariae. In ferrets necropsied following two intravenous injections of microfilariae, the majority of ferrets examined within 10 days after clearance of microfilariae had visible liver lesions to microfilariae identical to those of the ferrets infected with third-stage larvae; immediate cutaneous hypersensitivity and reaginic antibody were not consistently detected in ferrets injected with microfilariae. Sera from ferrets that had cleared circulating microfilariae were transferred passively into ferrets made microfilaremic by intravenous injection of microfilariae. Sera with microfilarial sheath-reactive IgG antibody titers (greater than or equal to 1:200) and microfilarial agglutination titers (greater than or equal to 1:40) rapidly cleared injected microfilariae (less than 24 hr); this serum also cleared or greatly reduced circulating microfilariae established by an infection with third-stage larvae; only the IgG-containing fraction of the sera was active in immune clearance. Sera that cleared microfilariae of B. malayi did not clear circulating microfilariae of Dirofilaria immitis or prevent recurrence of circulating microfilariae of B. malayi in ferrets infected with adult filariae.(ABSTRACT TRUNCATED AT 400 WORDS)     

Diethylcarbamazine activity against Brugia malayi microfilariae is dependent on inducible nitric-oxide synthase and the cyclooxygenase pathway

Background

Diethylcarbamazine (DEC) has been used for many years in the treatment of human lymphatic filariasis. Its mode of action is not well understood, but it is known to interact with the arachidonic acid pathway. Here we have investigated the contribution of the nitric oxide and cyclooxygenase (COX) pathways to the activity of DEC against B. malayi microfilariae in mice.

Methods

B. malayi microfilariae were injected intravenously into mice and parasitaemia was measured 24 hours later. DEC was then administered to BALB/c mice with and without pre-treatment with indomethacin or dexamethasone and the parasitaemia monitored. To investigate a role for inducible nitric oxide in DEC's activity, DEC and ivermectin were administered to microfilaraemic iNOS^-/- mice and their background strain (129/SV). Western blot analysis was used to determine any effect of DEC on the production of COX and inducible nitric-oxide synthase (iNOS) proteins.

Results

DEC administered alone to BALB/c mice resulted in a rapid and profound reduction in circulating microfilariae within five minutes of treatment. Microfilarial levels began to recover after 24 hours and returned to near pre-treatment levels two weeks later, suggesting that the sequestration of microfilariae occurs independently of parasite killing. Pre-treatment of animals with dexamethasone or indomethacin reduced DEC's efficacy by almost 90% or 56%, respectively, supporting a role for the arachidonic acid and cyclooxygenase pathways in vivo. Furthermore, experiments showed that treatment with DEC results in a reduction in the amount of COX-1 protein in peritoneal exudate cells. Additionally, in iNOS^-/- mice infected with B. malayi microfilariae, DEC showed no activity, whereas the efficacy of another antifilarial drug, ivermectin, was unaffected.

Conclusion

These results confirm the important role of the arachidonic acid metabolic pathway in DEC's mechanism of action in vivo and show that in addition to its effects on the 5-lipoxygenase pathway, it targets the cyclooxygenase pathway and COX-1. Moreover, we show for the first time that inducible nitric oxide is essential for the rapid sequestration of microfilariae by DEC.     

Galectin-9 induces maturation of human monocyte-derived dendritic cells

Maturation of dendritic cells (DCs) is critical for initiation of immune responses and is regulated by various stimulatory signals. We assessed the role of galectin (Gal)-9 in DC maturation. Culture of immature DCs with exogenous Gal-9 markedly increased the surface expression of CD40, CD54, CD80, CD83, CD86, and HLA-DR in a dose-dependent manner, although Gal-9 had no or little effect on differentiation of human monocytes into immature DCs. Gal-9-treated DCs secreted IL-12 but not IL-10, and they elicited the production of Th1 cytokines (IFN-gamma and IL-2) but not that of the Th2 cytokines (IL-4 and IL-5) by allogeneic CD4+ T cells. These effects of Gal-9 on immature DCs were not essentially dependent on its lectin properties, given that they were inhibited only slightly by lactose. We further found that a Gal-9 mutant that lacks beta-galactoside binding activity reproduced the above activities and that an anti-Gal-9 mAb suppressed them. Gal-9 induced phosphorylation of the MAPK p38 and ERK1/2 in DCs, and an inhibitor of p38 signaling, but not inhibitors of signaling by either ERK1/2 or PI3K, blocked Gal-9-induced up-regulation of costimulatory molecule expression and IL-12 production. These findings suggest that Gal-9 plays a role not only in innate immunity but also in acquired immunity by inducing DC maturation and promoting Th1 immune responses.     

Transfection of human monocyte-derived dendritic cells with CpG oligonucleotides

Monocyte-derived dendritic cells (mDC), the most frequently applied DC subset in clinical studies, which can be obtained easily from peripheral blood monocytes after incubation with GM-CSF and IL-4, have not been clearly demonstrated to be activated by CpG oligodeoxynucleotides (ODN). The development of novel molecular strategies - such as the use of CpG-ODN - to increase immunological functions and thus improve the therapeutic efficiency of mDC vaccines in the treatment of malignant diseases is highly desirable. CpG-ODN need to be internalized into specific intracellular compartments to be active. Therefore, we applied electroporation and lipofection and compared these techniques with incubation to overcome possible defects in localization. Conditions of CpG-ODN transfection of these cells were optimized using fluorescein-marked ODN 2216. We were able to achieve high transfection efficiencies with various methods of delivery. However, we did not observe increased expression of maturation-associated and functionally relevant surface antigens (CD14, HLA-DR, CD40, CD83, CD80 and CD86), significant secretion of IL-12 and IFN-alpha in culture supernatant, or enhanced antitumour activation of cytokine-induced killer cells. In conclusion, our results show that non-viral transfection of CpG-ODN is not sufficient to overcome resistance of mDC to CpG activation.     

Butyrate inhibits functional differentiation of human monocyte-derived dendritic cells

Dendritic cells (DCs) play a key role in immune function through antigen presentation by MHC and CD1, as well as cytokine production that shapes the immune response. Here we report that butyrate, a histone deacetylase inhibitor, inhibits the functional differentiation of human monocyte-derived DCs. Mature DCs were generated from monocytes in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), followed by 2 day LPS stimulation. Butyrate treatment throughout the culture period inhibited the expression of CD1 molecules, but not on CD83, CD86, and MHC molecules. The suppression was exerted at protein and mRNA levels. Butyrate-treated immature DCs also showed decreased expression of CD1 molecules. Moreover the butyrate-treated immature DCs showed lower production of IL-12 p40 and IL-6 in response to lipopolysaccharides and induced less Th1 cells in allogenic mixed lymphocyte reactions. Our results imply that histone acetylation is involved in regulating immune responses through regulating functional differentiation of DC. Thus HDAC may be one of the targets for controlling the immune response.     

Brugia malayi microfilaraemia in mice: a model for the study of the host response to microfilariae

Microfilariae of Brugia malayi were obtained from the peritoneal cavities of infected gerbils and were then injected intravenously into mice. A sub-periodic, nocturnal microfilaraemia was produced. The level of microfilaraemia was proportional to the number of parasites injected, with approximately 1-3% of microfilariae being found in the peripheral circulation. The duration of microfilaraemia was proportional to the number of parasites injected; it subsided by 30 days after injection of 104 microfilariae but was still present at a low level 120 days after injection of 2 x 105 microfilariae. A transient splenomegaly developed after injection of microfilariae. Histopathological examination revealed large numbers of microfilariae free in the lumens of pulmonary small blood vessels and without any accompanying inflammatory reaction. Lesser numbers of microfilariae were seen in the cardiac blood and hepatic and renal blood vessels for the first few days after injection. There was cellular proliferation in the splenic white pulp and vascular congestion of the red pulp. Microfilariae labelled with 51Cr were injected intravenously; 57% of radioactivity was found in the lungs, 8.5% in the liver and 2.9% in the spleen. Mice developed immediate hypersensitivity reactions to B. malayi antigen by 4 weeks after injection, but Arthus and delayed hypersensitivity reactions were not seen at any time. when mice which had been injected 5 months previously were challenged with a 2nd injection of microfilariae, there was an accelerated clearance of parasites over 2 weeks and a marked peripheral blood eosinophilia developed. In contrast with natural infections, in which the continuous production of microfilariae complicates assessment, this model provides a system in which factors controlling the circulation of microfilariae in the bloodstream can be studied independently.