Ann C. Vickery

THE LYMPHATIC PATHOLOGY OF BRUGIA PAHANGI IN NUDE (ATHYMIC) AND THYMIC MICE C3H/HeN

The nude (congenitally athymic) mouse, C3H/HeN is highly susceptible to infection with Brugia pahangi (Nematoda: Filarioidea). Normal, hairy mice show a strong thymus-dependent resistance and usually terminate the infection in the larval stages. The present study examined chronological histopathologic changes in the lumbar lymph nodes and adjacent lymphatic vessels of both hosts. In thymic mice, lymphangitis and perilymphangitis reached a maximum 14 to 17 days PI, about the time of disappearance of live worms. The infiltrate showed characteristics of both acute and chronic inflammation: eosinophils, neutrophils, eosinophilic precipitates, and sometimes necrotizing lymphangitis, as well as macrophages and plasma cells. The cellular infiltrate in nude mice was weaker and developed more slowly. Inflammatory responses to identifiable dead worms were seen in both types of hosts but appeared more frequently in thymic mice. Although variable in both models, the granulomas of thymic mice generally showed more tendency to cavitation, greater macrophage or epithelioid cell infiltration, more granulocytes, and appeared to be more destructive than the foreign body responses of nude mice. Whereas lymphangiectasis was generally progressive in nude mice, it was arrested before the end of the third week in thymic mice. In thymic mice, at maximum lumbar lymph node size (17 days), there were large areas of lymphocyte hyperplasia and heavy infiltration of plasma cells. Most nodes returned to normal mean size by the end of the second month. Little or no reactivity was seen in athymic mouse nodes. Our results suggest that some lesions of lymphatic filariasis are potentially thymus-independent: lymphatic fibrosis, lymphangiectasis, accumulations of macrophages and giant cells around disintegrating worms, calcification of worms, intralymphatic thrombosis, and moderate vascular infiltrates including eosinophils.

Histopathology of Brugia malayi-infected nude mice after immune-reconstitution

Long term (greater than 200 day) Brugia malayi-infected nude mice with grossly dilated lymphatics were reconstituted with 10(8) primed spleen cells from heterozygous donors. Histological and ultrastructural examination at 7, 14, 21 and 28 days post-reconstitution revealed progressive fibrosis, obliterative lymph thrombus formation, interstitial infiltrates and extensive perilymphangitis. Formation of lymph thrombi/granulomas was associated with killing of adult worms and microfilariae, and the predominant cell types involved were large granular macrophages. Langhans giant cells and eosinophils. Thus, the ability to initiate the formation of obstructive lesions in the dilated lymphatics of chronically parasitized nude mice by immunological reconstitution, suggests that several complex mechanisms might operate in stages to cause filarial elephantiasis.

Brugia malayi and Acanthocheilonema viteae: antifilarial activity of transglutaminase inhibitors in vitro.

The possible involvement of transglutaminase-catalyzed reactions in survival of adult worms, microfilariae (mf), and infective larvae of the filarial parasite Brugia malayi was studied in vitro by using the specific pseudosubstrate monodansylcadaverine (MDC) and the active-site inhibitors cystamine or iodoacetamide. These inhibitors significantly inhibited parasite mobility in a dose-dependent manner. This inhibition was associated with irreversible biochemical lesions followed by filarial death. A structurally related, inactive analog of MDC, dimethyldansylcadaverine, did not affect the mobility or survival of the parasites. Adult worms failed to release mf when they were incubated in the presence of MDC or cystamine, and this inhibitory effect on mf release was concentration dependent. Similar embryostatic and macrofilaricidal effects of MDC were observed in Acanthocheilonema viteae adult worms. These studies suggest that transglutaminase-catalyzed reactions may play an important role in the growth, development, and survival of filarial parasites.

Identification of a novel transglutaminase from the filarial parasite Brugia malayi andits role in growth and development

Recently, we reported the presence of a putative transglutaminase in adult female worms of Brugia malayi [1]. The enzyme activity was shown to be essential for in utero growth and development of microfilariae. Here, we demonstrate that adult worms of B. malayi have a large amount of epsilon-(gamma-glutamyl)lysine isopeptide bonds, a product of physiologically active transglutaminase. A 25-kDa immunoreactive band detected in female worm extracts by a monospecific monoclonal antibody (CUB 7401) against guinea pig liver transglutaminase was associated with the enzymatic activity. Unlike the mammalian enzyme, the parasite enzyme did not require Ca2+ for its catalytic activity. Furthermore, in utero developing embryos, especially during early stages of development, contained very high amounts of this enzyme. Adult female worms contained several proteins that could serve as suitable substrates for the enzyme. Inhibition of the enzyme activity by an enzyme-specific pseudosubstrate, monodansylcadaverine, led to a time- and dose-dependent inhibition of microfilariae production and release by gravid female worms. The inhibition of microfilariae production was due to the inhibition of transglutaminase-catalyzed crosslinking of parasite proteins that in turn seemed to be essential for in utero growth and development of the embryos. The results suggest that transglutaminase-catalyzed reactions may play an important role during early development of embryos to mature microfilariae inside the adult female worms of filarial parasites.

Significance of transglutaminase-catalyzed reactions in growth and development of filarial parasite, Brugia malayi

A novel form of transglutaminase enzyme [EC 2.3.2. 13] was identified in adult worms of Brugia malayi. The molecular size of this enzyme was 22-kilodaltons as determined by Western blot and immunoprecipitation, using a monoclonal (CUB 7401) or polyclonal antibodies against guinea-pig liver tissue transglutaminase. The enzyme was present in female worms only; adult males contained no detectable levels of the enzyme peptide. Possible involvement of transglutaminase-catalyzed reactions in growth and survival of filarial parasites was studied by using various enzyme-specific pseudosubstrates. Presence of these inhibitors resulted into a significant inhibition of microfilariae production and release by gravid female worms in a dose-dependent manner. These results suggest that transglutaminase-catalyzed reactions are essential for development of in utero growing embryos to mature microfilariae.

Regulatory cytokines in the lymphatic pathology of athymic mice infected with Brugia malayi

To investigate whether Brugia malayi-induced lymphatic inflammation is due to production of pro-inflammatory cytokines, we determined the lymph and serum levels of interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-alpha and granulocyte-macrophage colony stimulating factor (GM-CSF) using enzyme immunoassays. Serum from normal and infected mice did not show elevated cytokine concentrations. Samples of lymph from parasitized lymphatics had significantly increased levels of IL-1 (range = 6-1620 pg/ml), IL-6 (19-17,800 pg/ml), TNF-alpha (19-2000 pg/ml) and GM-CSF (4-275 pg/ml). The anti-inflammatory cytokine IL-4 (7-12 pg/ml) was in the normal range and no increase in interferon (INF)-gamma was detected in lymph samples. The data suggest that increased levels of mediators or cytokines localized in the lymphatics may be important contributors to massive lymphatic dilation and inflammation.

Effect of Brugia malayi on the growth and proliferation of endothelial cells in vitro.

Athymic mice (C3H/HeN) parasitized by Brugia malayi develop massively dilated lymphatics. The lymphatic endothelial lining is perturbed, and numerous mononuclear and giant cells are closely apposed to the endothelium. The hyperplastic endothelial cells and low opening pressure of the lymphatics suggest abnormal multiplication of these cells may be important in the dilation. We studied the in vitro growth rate of human umbilical vein endothelial cells cultured with adult worms and microfilariae of B. malayi. The tetrazolium salt reduction assays were used to quantify possible direct mitogenic or inhibitory effects. The growth factor-induced proliferation of endothelial cells was significantly suppressed by 44-51% on day 1, 46-81% on day 3, and 45-79% on day 5 in cultures containing adult female worms, which had greater suppressor activity on endothelial cell proliferation than male worms, microfilariae, or soluble adult worm extract. Culture supernatant containing female worm excretory-secretory products significantly inhibited the growth and multiplication of cells, suggesting that adult female worms release antigens or proteins that have inhibitory activity on growth factors necessary for endothelial cell proliferation in vitro. Excess human recombinant epidermal growth factor and bovine brain extract partly reversed the inhibitory activity of worms in culture and restored the endothelial cell proliferation when incubated with worm culture supernatant. Indomethacin and BW 775Hcl failed to restore normal endothelial proliferation in the presence of female worms, suggesting that parasite-derived prostanoids and cyclooxygenase products did not cause the inhibition. Lymph from dilated lymphatics, but not serum from infected mice, increased the proliferation of cells in vitro. Together, these data demonstrate that excretory-secretory products of B. malayi parasites suppress vascular endothelial proliferation in vitro. Furthermore, increases in the number of these cells in vitro in the presence of lymph suggest that parasite-induced host factors may be important in modulating the degree of proliferation.

Effect of carrageenan on the resistance of congenitally athymic nude and normal BALB/c mice to infective larvae ofBrugia malayi

Resistance of BALB/c mice to infective thirdstage larvae (L3) of the human filarial parasiteBrugia malayi is thymus-dependent, although the actual, effector mechanisms that mediate larval killing are unknown. The present study examined the effect of carrageenan (CGN) on the mechanisms of resistance toB. malayi infection in heterozygous (nu/+) and nude (nu/nu) mice. Mice were treated with CGN at a single dose of 20 or 200 mg/kg and were inoculated intraperitoneally 1 day later with 100 L3. The results showed a dose-dependent increase in the numbers of L4 and L5 that were recovered from nu/+ and nu/nu mice. CGN treatment also enhanced the recovery of mature adult worms from nu/nu mice and appeared to abolish partially the dichotomy of resistance between the usually more susceptible male and the more resistant female nu/nu mouse. Microfilariae were found in the peripheral blood and the peritoneal cavity of CGN-treated male and female nu/nu mice and in the peritoneal cavity of male but not female nu/+ mice. Fewer larval granulomas were recovered from the peritoneal cavity of treated mice. CGN-treated, parasitized nu/+ and nu/nu mice showed high titers of IgM and IgG antibodies. An experimental compound, CGP 20376, showed 100% larvicidal activity following the administration of a single dose of 20mg/kg to CGN-treated mice. From this study, we conclude that macrophages alone or in conjunction with other cells are actively involved in the resistance of mice toB. malayi L3.

Immunity to Brugia pahangi in athymic nude and normal mice: eosinophilia, antibody and hypersensitivity responses

Summary Congenitally athymic nude (nu/nu) mice, immunologically reconstituted by thymus grafting before inoculation with infective larvae, and mice heterozygous for the nu gene (nu/+), mounted potent protective humoral and cellular immune responses to Brugia pahangi. Although responses were not identical, both groups of mice produced IgM, IgG and IgE antibodies specific for adult worm antigen (S‐Ag) present in a crude aqueous extract, made immediate and delayed hypersensitivity footpad swelling responses when challenged with S‐Ag and eliminated their infection in the early larval stages. Heterozygotes also exhibited a marked eosinophilia which peaked coincident with larval killing. In contrast, thymus grafting of patent nudes had no effect upon microfilaraemias or adult worm burdens and did not completely protect against a challenge larval inoculum although antibodies specific for S‐Ag were produced. With the occasional exceptions of moderate immediate footpad swelling and very low titres of IgM specific for S‐Ag, no specific immune responses to B. pahangi were found in ungrafted nude mice which allowed full development of adult worms and supported patent infections.

Evaluation of different medium supplements for in vitro cultivation of Brugia malayi third-stage larvae

Growth and development of Brugia malayi (Nematoda: Filarioidea) third-stage larvae (L3) were compared in 5 medium supplements. The basic culture medium (NI) consisted of a 1:1 (v/v) mixture of NCTC-135 and Iscoves modified Dulbeccos medium, an antibiotic/antimycotic mixture, and 1 of the following 5 supplements: 25 mg/ml bovine albumin fraction-V (BAF), 10% fetal bovine serum (FBS), 10% commercially obtained human serum (CHS), 10-15% pooled human serum from hospital patients (PHS), and 10-15% human serum from a single individual (SHS). Cultures were maintained at 37 C in an atmosphere of 5% CO2 in air. NI-BAF and NI-CHS did not support molting of L3 to fourth-stage larvae (L4), whereas NI-FBS, NI-PHS, and NI-SHS did support molting of L3 to L4 but only the larvae in NI-SHS attempted the fourth molt. Growth and development of in vitro larvae in NI-PHS and NI-SHS were comparable to that observed in jirds for the first 28 days, after which the in vitro larvae lagged behind in vivo larvae. Optimal growth and development may be dependent on certain as yet unidentified components of specific human serum.