Jeffrey F. Williams

Ivermectin distribution in the plasma and tissues of patients infected with Onchocerca volvulus

Objective:To determine the distribution of ivermectin in plasma and tissues of onchocerciasis patients following a single oral dose of 150 μg kg−1.Setting:Medical Department at Soba University Hospital, Khartoum.Patients:Twenty five patients and fourteen healthy volunteers.Methods:Serial blood samples were obtained from both groups. Tissue samples were removed from various patients as full thickness skin punch biopsies or during nodulectomy. Ivermectin concentration was determined by radioimmunoassay.Results:The plasma pharmacokinetic variables for patients were; maximum plasma concentration 52.0 ng ml−1; time to achieve maximum concentration, 5.2 h.; elimination half life, 35.0 h; and the area under the plasma concentration curve versus time, 2852 ng⋅h ml−1. In healthy volunteers, the plasma ivermectin distribution was similar to that in patients, and both groups showed a tendency for a second rise in plasma concentration of the drug suggestive of enterohepatic recirculation. Ivermectin was detected in tissues obtained from patients. Fat showed the highest and most persistent levels, whilst values for skin, nodular tissues, and worms were comparable. Subcutaneous fascia contained the lowest concentrations.Conclusion:Infection with O. volvulus does not affect the pharmacokinetics of ivermectin, and filarial infected tissues and parasites themselves do take up the drug. There may be prolonged retention of ivermectin because of depot formation in fat tissue.

TAENIA TAENIAEFORMIS (CESTODA) IN THE RAT: ULTRASTRUCTURE OF THE HOST-PARASITE INTERFACE ON DAYS 8 TO 22 POSTINFECTION

The host-parasite interface was examined at the ultrastructural level 8 to 22 days postinfection (DPI) with metacestodes of Taenia taeniaeformis in the rat. Throughout this phase of development the parasite surface was invested with a dense surface coat of complex microtriches. At 8 to 14 DPI the plasma membrane of each microthrix extended beyond the distal end of the electron-dense tip, forming a slender tubular streamer over 10 microns long; by 18 DPI these had shortened and withered. Host cell processes interdigitated with the microtriches without evidence of harm to the parasite surface or the underlying tegument. The cells, on the other hand, became damaged, and their contents were shed into the matrix surrounding the microtriches. Lipid inclusions appeared within the parasites, and in the cytoplasm of surrounding inflammatory cells. By 22 DPI fibroblastic activity had resulted occasionally in the formation of a capsule surrounding a free-floating cysticercus, while in others intense granulocytic infiltration persisted with abutment and intermeshing of host cell and parasite surface processes; however there was still no evidence of any adverse effect on the microtriches, though many granulocytes were clearly pyknotic and degenerating. Evidently, the vigorous cellular response of the host is ineffective in either containing the expansion of the parasite or compromising the integrity of its surface membrane. The changing characteristics of the microtriches may be related to the need for dissolution of both intercellular matrices, and host cells as the vesicular organism rapidly increases in volume.

Dirofilaria immitis: Do filarial cyclooxygenase products depress endothelium-dependent relaxation in the in vitro rat aorta?

Endothelial cells modulate the function of their underlying smooth muscle. Thus, altered endothelial behavior could be important in the pathogenesis of vascular and lymphatic diseases, including human and animal filariasis. Endothelium-dependent relaxation is depressed in both in vivo canine femoral artery of dogs infected with Dirofilaria immitis and in vitro rat aorta exposed to adult D. immitis. The experiments reported here were designed to determine if filarial cyclooxygenase products could depress endothelium-dependent relaxation in vitro. Pretreatment of the parasites, but not the vascular ring, with either indomethacin or aspirin, prevented filarial-induced depression of relaxation. Analysis of heartworm-conditioned medium by gas chromatography--mass spectrometry revealed two peaks in the biologically active medium that were not present in the control. One peak had a retention time and chromatographic profile characteristic of derivatized PGD2 standard, and the other was not identified. Incubation of the vascular ring with PGD2 mimicked filarial-induced depression of endothelium-dependent relaxation at low, but not high, concentrations of acetylcholine. Thus, filarial PGD2 may be involved in altered endothelium-dependent relaxation seen in heartworm-infected dogs.

Interactions between taenia taeniaeformis and host cells in vitro: rapid adherence of peritoneal cells to strobilocerci.

Abstract Engelkirk P. G ., Williams J. F. and Signs M. M. 1981. Interactions between Taenia taeniaeformis and host cells in vitro : rapid adherence of peritoneal cells to strobilocerci. International Journal for Parasitology 11 : 463–474. Strobilocerci of Taenia taeniaeformis , incubated for l h in vitro with various combinations of serum and peritoneal cells from infected or non-infected rats, were examined at the ultrastructural level for evidence of cell adherence and tegumental damage. Maximal adherence and surface alterations occurred when larvae were incubated in the presence of cells and fresh serum. This was true regardless of whether the cells or the serum had been obtained from infected or non-infected donors. No tegumental damage was seen when parasites were incubated with or without cells in the absence of serum. Serum enhancement was either much reduced or abolished by heat treatment (56°C for 1 h). In the presence of EDTA, tegumental lesions still developed, but adherence of cells, especially those from non-infected rats, decreased markedly. The predominant cells interacting with the larval surface were eosinophils; these took up parasite material within phagosomes and appeared to strip microtriches from the tegumental free surface. Mast cells, some of which became degranulated, were also present in the adherent cell masses. The results indicate that potent non-specific effector mechanisms can rapidly damage the tegument of T. taeniaeformis, in vitro , in contrast to the failure of recognition and rejection by host defenses in vivo . Established strobilocerci are therefore not invulnerable but the balance of the host-parasite relationship in vivo must favor their survival.

A Simplified Method for Staining Mast Cells with Astra Blue

The copper phthalocyanin dye astra blue has been used to stain differentially mast cells of the intestine; however; the procedure has not been used widely because of the difficulty in preparing and using the dye solution. Described here is a simple, reliable, and consistent method for selectively staining mast cells using a dye solution that may be prepared in any laboratory without the aid of sophisticated pH metering equipment. Astra blue is mixed with an alcoholic solution containing MgCl2-6H2O and the pH indicator pararosaniline hydrochloride. Concentrated hydrochloric acid is added dropwise, changing the dye mixture from purple to violet and then to blue. In this low range the weakly ionizing ethanol provides a more stable hydrogen ion concentration than the corresponding aqueous solutions used previously. Alcoholic acid fuchsin is a convenient counterstain, and this simple procedure then provides good contrast between the blue staining mast cell granules and the red tissue background.

Ivermectin treatment in severe asymmetric reactive onchodermatitis (sowda) in Sudan

Ivermectin efficacy and post-treatment reactions in asymmetric severe reactive ochodermatitis (sowda) were studied in 8 patients with sowda syndrome and 6 with mild generalized onchodermatitis in Sudan. Initial skin snips from 12 patients contained microfilariae (1-9 per mg skin). Patients were treated in hospital with a single oral dose of c. 150 micrograms/kg ivermectin (103-200 micrograms/kg) and monitored for frequency and severity of post-treatment reactions for 4 weeks. Serial samples of heparinized blood were collected over the first 24 h after treatment for determination of ivermectin pharmacokinetics. Skin snips from all patients on days 3 and 28 revealed no microfilariae. Post-treatment reactions were more common and severe in individuals with sowda; they consisted mainly of musculoskeletal pain, local swellings with pitting oedema, and lymph gland tenderness and enlargement. No relation was established between these reactions, the microfilarial infection intensity, or the plasma pharmacokinetic profiles. A single oral dose of ivermectin cleared the skin of microfilariae and led to improvement of symptoms and dermatological signs of sowda, but resulted in more marked reactions than in cases of generalized onchodermatitis.

Taenia hydatigena and T. ovis: Antigen sharing: XII. Immunological responses of the mammalian host against tapeworm infections☆

Single large doses of the eggs of Taenia hydatigena and T. ovis were fed to lambs either simultaneously or separated by an interval of 3 months. The proportion of the cysticerci of T. hydatigena which failed to survive was greater than expected when the eggs of T. ovis were fed prior to or simultaneously with those of T. hydatigena. In contrast, the survival rate of cysticerci of T. ovis was enhanced when the eggs of T. hydatigena were fed before but not simultaneously with those of T. ovis. A working hypothesis based on variations in the conformation of the antigens on the wall of the bladder is proposed to explain the differential survival of larval tapeworms during the first 3 months of development in reciprocal heterologous tapeworm infections.

Lipid composition of metacestodes of Taenia taeniaeformis and lipid changes during growth.

A lipid analysis was performed on developing metacestodes of Taenia taeniaeformis removed from the livers of rats at times varying from 3 to 35 weeks post infection. Lipid accounted for 7-21% of the dry weight of the parasites. The highest proportions were found at the earlier stages. The distribution was as follows; neutral lipid 27-45%; glycolipid 5-11%; and phospholipid 50-61%. The major neutral lipid was cholesterol, and minor neutral lipids were sterol esters, triglycerides, diglycerides and monoglycerides. Hydrocarbons were present throughout development, but in the highest amounts at the earlier stages. Five different glycolipids were found, all of which were identified as glycosphingolipids. An increase in the proportion of more complex glycolipids was noted as parasites grew older. Ten different phospholipids were identified, with the major components being phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. Other phospholipids were: lysophosphatides, phosphatidylinositol, phosphatidic acid, diphosphatidylglycerol, sphingomyelin, and an unknown phospholipid component. Changes in the relative amounts of the two major phospholipids were found when the early and late stages were compared. Two lipids found throughout development were identified as glycosylated dolichol phosphates, and they comprised between 1 and 3% of the total phospholipid fraction. Nineteen fatty acids were detected, and the fatty acid distribution for each lipid class at each stage was determined. Seven major fatty acids were common to each. These were: hexadecanoic, octadecanoic, oleic, linoleic, arachidonic, docosanoic, and docosahexaenoic.

Taenia taeniaeformis: Gastrointestinal hyperplasia in chronically infected rats

Abstract Hyperplastic pathological changes in the gastrointestinal tract and serum gastrin levels were measured at postmortem in rats given long-term infections with metacestodes of Taenia taeniaeformis. Stomach weights were greater than in controls from the beginning of the experiment at 63 days postinfection (DPI) onward, and generally reached highest levels (>16 g) in the more chronically infected rats (up to 368 DPI). Gross and histological evidence of papillary hyperplasia of gastric mucosal cells increased with duration of infection. Intestinal weights also increased with time, but to a much lesser degree than those of the stomachs. These changes were sustained throughout the period of study, and were associated with significant elongation of villi and increased mucosal crypt depth, especially in the duodenum. In the most chronically infected rats multiple cystic dilatations of crypts occurred, and within them accumulated masses of mucus and necrotic cells often became calcified. Villi, sometimes over 1 mm in length, became club-like and frequently fused together. Mucosal mast cell (MMC) numbers in the small intestine were significantly higher than in controls throughout infection. Hyperplastic changes were detected inconsistently in the colonic mucosa, but there were no effects on the esophagus. Splenomegaly was a constant finding in infected rats, which also showed an earlier onset of thymic atrophy than normal age-matched controls. Serum gastrin levels were elevated in all affected rats at 5 months postinfection, but by the end of the first year some animals had normal gastrin levels, even though they showed severe hyperplastic gastroenteropathy at necropsy. Attempts to induce changes in gastric and intestinal weights, MMC numbers, or serum gastrin by the serial inoculation of extracts or in vitro products of T. taeniaeformis strobilocerci were unsuccessful. Surgical implantation of strobilocerci intraperitoneally also produced no measurable changes in these parameters. The results suggest that the hyperplastic stimulus provided by T. taeniaeformis is sustained for many months, and that there is a gradient of responsiveness in gastrointestinal tissues, declining from the glandular stomach mucosa posteriorly. The stimulus may be augmented by endogenous gastrin, but this hormone is not necessary for maintenance of hyperplastic changes. Our failure to induce changes in vivo artificially suggests that more subtle criteria, perhaps involving in vitro influences on gastric or intestinal cell turnover, will be necessary to establish if the parasite exerts its effects on host cells directly or indirectly.

Immunological response of the rat to infection with Taenia taeniaeformis: Protective antibody response to implanted parasites

Abstract Intraperitoneally implanted metacestodes of either T. taeniaeformis or T. crassiceps in rats provoked a high degree of resistance to oral challenge with eggs of T. taeniaeformis . This resistance was passively transferred to normal recipients with serum. Immunoglobulin fractions of immune serum containing IgG 1 or IgM were most effective in passive transfer and little activity was associated with IgG 2 antibodies. No skin-sensitizing antibodies were detectable in immune sera. These findings are in sharp contrast to previous observations involving protective immunoglobulins and reaginic antibodies in serum from rats with hepatic cysticerci of T. taeniaeformis. Possible reasons for this are discussed. Cysticerci implanted into normal rats survived for at least 21 days with no sign of host rejection, whereas those implanted into rats with hepatic infections with T. taeniaeformis were killed and encapsulated. Similar results were obtained by implanting cysticerci in normal rats given inoculations of complete Freunds adjuvant. Repeated inoculations of immune serum had no effect on the survival of implanted cysticerci, and it was concluded that exposure to infection by oncospheres provokes cellular defense mechanisms which can be effective against cysticerci in abnormal sites. Why these mechanisms are inoperative against hepatic cysticerci remains unclear.