Immunity in taeniasis-cysticercosis I. Vaccination against Taenia taeniaeformis in rats using purified antigen.

Artificial immunization of rats against Taenia taeniaeformis was studied using somatic antigen (Som-Ag) and excretory-secretory antigen (ES-Ag). It was found that both Som-Ag and ES-Ag stimulated immediate-type hypersensitivity and delayed-type hypersensitivity reactions of similar levels. Antibody levels rose from the 2nd wk and peaked around the 6th and 7th wk. Both IgM and IgG were detectable from the 2nd wk onwards, with IgG at a considerably higher level compared to IgM. It terms of protection, 90-100% reduction in cyst counts were detected if the rats were challenged 10 days or more after immunization. In all cases, no significant difference was observed between immunization with either Som-Ag or ES-Ag were purified and characterized using Sephadex G-200 chromatography, double immunodiffusion, and disk acrylamide gel electrophoresis. A purified antigen (mol wt, 140,000 daltons) was obtained, and highly significant protection against infection resulted with injections of 50, 10, or 1 mug doses of this antigen with complete Freund's adjuvant.

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.

A direct fluorescence technique for the rapid detection of sheathed microfilariae in blood smears.

Thick and thin blood smears containing microfilariae of Wuchereria bancrofti, Loa loa, Brugia malayi, Brugia pahangi, Brugia patei or Acanthocheilonema viteae were prepared from either cryopreserved blood samples or from freshly collected blood, fixed in methanol and treated with a fluoresceinated lectin wheat germ agglutinin. Sheathed microfilariae of W. bancrofti, L. loa, B. malayi, B. pahangi and B. patei in the blood smears could be easily detected and counted using a fluorescence assay. The unsheathed microfilaria of Acanthocheilonema viteae did not fluoresce. The possibility of adapting this technique, which does not require the use of parasite specific antibody for the sensitive, parasitological detection of filarial infections, is discussed.

Lectin binding to larval stages of Brugia patei.

Surface properties of microfilariae (mf) and infective larvae of Brugia patei were investigated to compare them to previous studies with the other brugian species. Of all the lectins tested, only wheat germ agglutinin (WGA) binds to the sheath surface of mf indicating the presence of N-acetyl-D-glucosamine as a major surface carbohydrate. However, cuticle of infective larvae failed to show binding of these lectins. Enzyme treatment of mf with N-acetyl-D-glucosaminidase and L-fucosidase has exposed D-mannose, N-acetyl-D-galactosamine and L-fucose on the sheath surface. The binding of lectins to intact mf and to enzyme-treated mf appeared to be specific as pretreatment with specific inhibitory sugars completely abolished the binding activity. This is the first study conducted with this filarial parasite and it established that B. patei is similar to other species of Brugia but differs from Wuchereria in its surface-lectin binding properties.

Effects of serum from treated patients on antibody-dependent cell adherence to the infective larvae of Brugia malayi.

Adherence assays were used to demonstrate the in vitro effect of serum-dependent cellular adherence of human buffy coat cells to infective larvae of Brugia malayi in filariasis patients treated with antifilarial drugs. In this study, microfilaraemic patients were treated with either diethylcarbamazine citrate (DEC), mebendazole or levamisole hydrochloride. It was found that DEC and mebendazole decreased the motility of infective larvae due to a direct action of the drugs. Sera of levamisole-treated patients caused increased adherence of human buffy coat cells to infective larvae, leading to a decrease in motility and cuticular damage as confirmed by scanning electron microscopic studies. However, serum of levamisole-treated patients alone could cause a similar lethal effect on infective larvae. Studies with the indirect fluorescent antibody test suggested that IgM was involved in this phenomenon. Complement did not appear to be important.

Brugia malayi: ivermectin inhibits the exsheathment of microfilariae.

Brugia malayi-infected microfilaremic jirds (Meriones unguiculatus) were treated with ivermectin at a single dose of 200 micrograms/kg of body weight injected subcutaneously. Susceptible Aedes aegypti mosquitoes were fed on treated jirds 24 hours later. Mosquitoes fed on untreated jirds served as controls. Infected mosquitoes were dissected at 1, 3, 24, 48, 72, and 96 hr after the blood meal, and differential counts of sheathed microfilariae, exsheathed microfilariae, and cast sheaths were performed using fluoresceinated wheat germ agglutinin. Microfilariae failed to exsheath in mosquitoes fed on ivermectin-treated jirds. Microfilariae from ivermectin-treated jirds also did not exsheath in vitro in the presence of 10 mM CaCl2, whereas 85-90% of sheathed microfilariae from untreated jirds exsheathed in vitro. In addition, sheathed microfilariae from untreated jirds, when pretreated in vitro with ivermectin at 0.25, 0.5, or 1 microgram/ml, lost their ability to exsheath in vitro in the presence of 10 mM CaCl2. However, ivermectin treatment had no effect on exsheathing of microfilariae when incubated with papaya protease. Thus, ivermectin appears to inhibit the intrinsic exsheathing process of microfilariae in the mosquito host, thereby blocking their development and further transmission of infection.

Specific depression of cell-mediated immunity in Malayan filariasis.

The possible depression of cell-mediated immunity by long-term Brugia malayi infection in jirds (Meriones unguiculatus) was investigated. Different groups of infected jirds were sensitized with dinitrofluorobenzene, sheep red blood cells, Dirofilaria immitis adult antigens and B. malayi adult antigens. The 24-hour delayed type hypersensitivity skin response to testing with antigen was measured as an in vivo correlate of cell-mediated immunity. The delayed-type hypersensitivity responses to dinitrofluorobenzene, sheep red blood cells and D. immitis antigens were normal but the response to B. malayi antigens was significantly depressed, confirming that long-term B. malayi infection depresses cell-mediated immunity and that this depression is specific to B. malayi antigens.

Studies on the experimental chemotherapy of Angiostrongylus malaysiensis infection in rats with mebendazole and levamisole.

The effect of levamisole hydrochloride and mebendazole on Angiostrongylus malaysiensis infection in albino rats was studied. Animals at different stages of infection were treated with various oral doses of levamisole and mebendazole with the aim of finding an effective treatment regime. Levamisole was most effective for treating rats seven days after infection but its efficacy dropped as infection progressed. Mebendazole given at a dose of 1 mg/kg for five days was more effective against early larval stages (97.39% efficacy). At 5 mg/kg for five days mebendazole was more effective than levamisole against all stages of the infection.

Immune responses in human Brugia malayi infections: serum dependent cell-mediated destruction of infective larvae in vitro.

Serum dependent cell-mediated cytotoxicity of subperiodic Brugia malayi infective larvae in vitro was investigated. In vitro cellular adherence of normal human buffy coat cells to infective larvae of B. malayi was promoted by sera from patients with elephantiasis, tropical pulmonary eosinophilia (TPE) and amicrofilaraemic symptomatic filariasis, as well as by sera from normal subjects from filariasis endemic areas. However, strongest adherence was observed with TPE sera. Scanning electron microscopic (SEM) studies confirmed that the cellular adherence resulted in gross surface damage to the infective larvae. Studies with the indirect fluorescent antibody test (IFAT) suggested that IgG and/or IgM might be involved in the process of adherence. Complement did not appear to be important.

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. Langhan's 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.

Observations of Breinlia booliati in a new host, Rattus rattus jalorensis, from Kuantan, state of Pahang, Malaysia.

Breinlia booliati Singh and Ho, 1973 is described from the Malaysian wood rat, Rattus rattus jalorensis Bonhote. The parasites presented here were originally discovered in 1955 in Kuantan, Malaysia, but were not classified until now. On the basis of morphological observations of anatomical structures and comparisons with other species of Breinlia, it was determined that the parasites were B. booliati. The parasites discussed here show slight deviation from B. booliati, but they do not warrant a new species classification. There is some variation in anatomical measurements, the number of male caudal papillae, and the morphology of the microfilariae. Breinlia booliati from a new host is described in this article, with a brief discussion on Rattus species that are hosts of B. booliati and vectors that transmit the parasite. The occurrence of B. booliati in R. r. jalorensis represents the first report of the parasite in this host.

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 Iscove's modified Dulbecco's 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.

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.

Cultured endothelial cells from lymphatics of nude mice parasitized by Brugia malayi.

Endothelial cells from dilated inguinal lymphatics of congenitally athymic nude mice, parasitized by adult Brugia malayi, were placed in culture. Cells formed a loose monolayer and exhibited a typical cobblestone appearance. When microfilariae were present in cultures, they frequently appeared to be attached to the monolayer by one end. Approximately 75% of the primary explant cells were positive for Factor VIII-associated antigen, comparable to bovine artery endothelial cells used as a control. With few exceptions, cultures were uncontaminated with fibroblasts or other non-endothelial cell types. Large granular cells with characteristics of mononuclear/macrophage cells appeared in long term and unpassaged cultures. Cells remained viable in culture for an average of 60 days, 5 to 6 passages, before becoming highly vacuolated and assuming a rounded configuration. Viability of the cells was dependent upon heparin, serum and endothelial cell growth factor. The propensity of the lymphatics of nude mice to become greatly dilated in the presence of viable adult worms of B. malayi will prove to be important not only for the study of the effects of the parasite and its products upon endothelial cells, but also because a source of murine lymphatic endothelial cells can be readily available for functional studies.

Chemotherapy and chemoprophylaxis of Angiostrongylus malaysiensis infection in rats with levamisole and mebendazole.

The chemoprophylactic and chemotherapeutic effects of levamisole and mebendazole on Angiostrongylus malaysiensis infection in rats were studied. Both drugs were ineffective in preventing infection while the post-infection treatment showed 100% efficacy. Furthermore, levamisole and mebendazole when given in combination appeared to have an antagonistic effect.

The role of cell-mediated immunity in Taenia taeniaeformis infections.

A functional cell mediated immunity (CMI) response was recorded in rats experimentally infected with Taenia taeniaeformis larvae. The presence of CMI was manifested in the delayed type hypersensitivity (DTH) reaction recorded between 12 hours and 24 hours on elicitation with antigen. The time course of the immediate type hypersensitivity (ITH) over 10 weeks showed 2 peaks around the 2nd and the 6th weeks of infection, whereas the DTH response was generally weaker and more uniform over the same time course. Transfer of peritoneal cells from infected rats conferred partial protection to normal recipient rats against a challenge infection. However, optimal protection was only about 50% with transfer of 0.625 X 10(7) cells/rat, and no increase in protection was possible even with transfer of higher cell concentrations.

Experiences in the management of chest injuries and a review of current management.

Eleven cases of chest trauma managed in the Intensive Care Unit (ICU), Alexandra Hospital were reviewed. Common manifestations were: rib fractures, haemothorax, pneumothorax, pulmonary contusion and flail chest. Nine patients had fractures on other sites of the body and three patients had associated abdominal injuries requiring laparotomy. Patients were referred to the ICU only when they were in respiratory distress. Transfer to the ICU occurred one to three days after admission to the hospital. Eight patients subsequently had to be ventilated. Two patients died. Respiratory failure in chest trauma is often the result of damage to the parenchyma, atelectasis and infection. Whilst the extent of parenchyma lung damage is dependent upon the severity of the injury and therefore not medically preventable, atelectasis and infection can be avoided. Patients with significant chest trauma should therefore be admitted directly to the Intensive Care Unit and the 'Expectant Therapy' instituted.