William L. Hanson

Improved therapy of experimental leishmaniasis by use of a liposome-encapsulated antimonial drug

Abstract The anti-leishmanial antimonial drug meglumine antimoniate (Glucantime ®) was incorporated into liposomes, and was tested for effects against experimental leishmanial infection in hamsters. When the hamsters had been infected for a short period before treatment (3 days), treatment doses of 416 mg/kg of free meglumine antimoniate, or 4 mg/kg of liposome-encapsulated drug, each gave 99.8% suppression of parasites. After 10 or 17 days of infection prior to treatment liposome-encapsulated compound was more than 300 times as effective as the antimonial drug alone. Use of liposomes containing appropriate drugs is proposed as a markedly superior means to treat certain chronic intracellular parasitic infections.

Testing of drugs for antileishmanial activity in golden hamsters infected with Leishmania donovani.

Abstract Hanson W. L. , Chapman W. L. Jr. and Kinnamon K. E. 1977. Testing of drugs for antileishmanial activity in golden hamsters infected with Leishmania donovani. International Journal for Parasitology7: 443–447. An existing procedure has been modified to make possible the screening of 10–15 selected compounds per week for suppressive activity against Leishmania donovani in a golden hamster model. Compounds with previously known antileishmanial activity were consistently found to have suppressive activity with this procedure. Antileishmanial activity is reported for the first time with compounds of the 6- and 7-aminoquinoline classes. The activity of all compounds studied is compared to that of Glucantime® which is the reference compound used in this procedure.

SUSCEPTIBILITY OF FIVE STRAINS OF MICE TO BABESIA MICROTI OF HUMAN ORIGIN

One outbred (CF1) and four inbred (BALB/c, C57, CBA and C3H) strains of mice were tested for susceptibility to Babesia microti of human origin. Of these, intact C3H mice developed higher parasitemia than all other intact mice, while BALB/c mice developed the highest parasitemia among splenectomized mice. Susceptibility was not related to H-2 haplotype in any obvious way. Because C3H and BALB/c mice developed relatively high initial peak parasitemias, the parasite was serially passaged in both of these mouse strains in an attempt to increase parasite virulence. After 30 passages in BALB/c and 49 passages in C3H mice over a period of 12 months, maximum parasitemias were 50 times higher than those observed initially. After the peak parasitemias of these two mouse-adapted parasites had stabilized, the relationship between onset and level of maximum parasitemia and number of parasites inoculated was determined. With both C3H- and BALB/c-adapted parasites, as inoculum size increased, the time required to reach maximum parasitemia decreased and the level of maximum parasitemia increased. Studies involving infection of either mouse strain with parasites adapted to the heterologous mouse strain indicated that C3H mice were more susceptible than BALB/c mice to homologous or heterologous parasites. These data suggest that the virulence of B. microti to the mouse can be increased by prolonged passage in this host. Once adaptation to this host species has occurred, virulence appears to be more dependent on the innate susceptibility of the mouse strain than on adaptation of the parasites to a particular strain of mouse.

Transfer of immunity to Babesia microti of human origin using T lymphocytes in mice.

Abstract Lymph node and spleen cells from mice infected with Babesia microti of human origin developed the ability to transfer adoptive immunity to naive mice within 25 days after infection. This protective activity was greater in cells obtained at 32 days than in cells obtained at 25 days postinfection and remained stable up to 52 days postinfection. Recipients of lymph node cells and spleen cells displayed similar peak parasitemias although 2 days after peak parasitemia, immune spleen cell recipients had significantly lower parasitemias than immune lymph node cell recipients. Strong protective activity was demonstrated when cells were transferred 1 day postinfection, while equal numbers of cells, transferred 3 days postinfection did not confer significant protection over nonimmune cells. There was also a suggestion that the number of immune spleen cells necessary for significant protection was directly related to the number of parasites inoculated. The subpopulation of lymphocytes responsible for the transfer of adoptive immunity to B. microti of human origin was then studied in BALB/c mice depleted of T lymphocytes by thymectomy and lethal irradiation. One day after infection with B. microti , T-cell-depleted mice were given complement-treated immune spleen cells, anti-θ serum-treated immune spleen cells, nonimmune spleen cells, or no cells. Similar experiments were performed comparing the effects of anti-immunoglobulin serum-treated and unfractionated immune spleen cells on B. microti parasitemia. Treatment with anti-θ serum abrogated the protective activity of immune spleen cells while anti-immunoglobulin serum treatment had no effect. These results suggest that immunologic memory of B. microti in BALB/c mice is modulated by T rather than B lymphocytes.

Liposomes in leishmaniasis: effects of parasite virulence on treatment of experimental leishmaniasis in hamsters

During studies on the use of liposomes as drug carriers in experimental leishmaniasis in hamsters, we noted incidentally that the apparent virulence of the infection often varied widely between different large groups of animals. When the death rates among control animals (injected only with saline) were compared with hepatic parasite counts of survivors in the same group, three distinctive types of infection were observed: type I, low death rate, low parasite count in survivors; type II, high death rate, low parasite count in survivors; type III, high death rate, high parasite count in survivors. The apparent virulence, based on death rates both at early and late stages of infection, was in the order I less than II less than III. Therapeutic efficacy of a drug (meglumine antimoniate) or liposome-encapsulated drug against each type of infection was in the order I greater than II greater than III. Liposomes reduced the drug dose required for each infection type many hundred-fold and reduced the death rate for type I to zero. However, among animals with type III (or even type II) infection certain individuals were completely refractory to treatment, even when liposome-encapsulated drug was employed, and the lowest mortality rate achieved was approximately 30%. This latter resistance to treatment may have been due to irreversible tissue damage caused by advanced disease, or it may have reflected resistance of certain virulent infections to treatment.

Leishmania donovani, Plasmodium berghei, Trypanosoma rhodesiense: antiprotozoal effects of some amidine types.

Abstract A series of 39 diamidines and cyclic congeners was investigated for antiprotozoal effects in standard animal models. The test systems employed were the following: Leishmania donovani in hamsters, Plasmodium berghei (trophozoite) in mice, and Trypanosoma rhodesiense in mice. None of the compounds was found to exhibit appreciable antimalaria or antileishmanial activity. One compound, 2,5-bis(4-guanylphenyl)furan dihydrochloride, was identified as having antitrypanosomal activity in the same range as pentamidine, and deemed worthy of further study.

Leishmania donovani: Cellular and humoral immune responses after primary and challenge infections in squirrel monkeys, Saimiri sciureus

Cellular and humoral immune responses were studied in squirrel monkeys after primary and challenge infection with a Khartoum strain (WR 378) of Leishmania donovani. Each of 7 squirrel monkeys, Saimiri sciureus, was inoculated intravenously with 5 X 10(7) amastigotes/kg body weight, and one other monkey (control) was inoculated with uninfected hamster spleen homogenate. Five infected monkeys recovered from visceral leishmaniasis and two infected monkeys died. Three of the five squirrel monkeys which recovered from the primary infection demonstrated acquired resistance when challenged with an intravenous inoculation of 1.0 X 10(8) amastigotes of L. donovani/kg of body weight. Each of these same three monkeys, the two remaining monkeys which recovered from the primary infection and an uninfected control monkey, were challenged subsequently with an intradermal injection of 2.2 X 10(7) promastigotes of L. braziliensis panamensis (WR539) and developed cutaneous lesions. The reactivity of peripheral blood leukocytes from infected squirrel monkeys to phytohemagglutinin was depressed 2 to 10 weeks after infection, and the reactivity to concanavalin A was not affected. Data on responses to pokeweed mitogen were inconclusive. Reactivity to leishmanial antigens was detected at 12 weeks after infection, which coincided with a marked decrease or disappearance of parasites in liver imprints. Two of five surviving squirrel monkeys developed weak delayed skin test responses to leishmanin antigens after 23 weeks; the three remaining monkeys were anergic during the primary infection but developed strong delayed skin test responses to leishmanin antigens at 7 weeks after a challenge with L. donovani. All squirrel monkeys inoculated with L. donovani developed a hyperproteinemia, hypergammaglobulinemia, hypoalbuminemia, and a reversal of the albumin/globulin ratio between 4 to 18 weeks after infection. Plasma IgM and IgG levels were increased between 2 to 18 weeks after infection; much of this increase was due to IgG. Class-specific antileishmanial antibodies, with generally low IgM and high IgG titers, reached a maximum after 14 and 16 weeks, respectively. A correlation was observed between concentration of gamma-globulins and plasma IgM and IgG levels, but not gamma-globulin concentrations and maximum titers of class-specific antileishmanial antibodies. Squirrel monkeys challenged with L. donovani again developed hyperproteinemia, hypergammaglobulinemia, and increased concentrations of plasma IgM and IgG which correlated with high titers of IgG class-specific antileishmanial antibody 4 weeks after reinoculation.(ABSTRACT TRUNCATED AT 400 WORDS)

Liposomal chemotherapy in visceral leishmaniasis: an ultrastructural study of an intracellular pathway.

The intracellular fate of liposomes administered intracardially was examined in the liver and spleen of hamsters experimentally infected withLeishmania donovani. Separate groups of animals were treated with liposomes containing either an antileishmanial agent, a colloidal gold marker, or saline. Ultrastructural examinations of lysosomal interactions with the parasitophorous vacuole and with phagocytized liposomes were made. Lysosomes readily fused with the parasitophorous vacuoles but appeared to have little effect on the parasite, possibly due to the production of enzyme inhibitors. Liposomes rapidly became localized in lysosomes subsequent to endocytosis by macrophages. Morphologic evidence suggested that secondary lysosomes containing liposomal residues then fused with the parasitophorous vacuole. Aspects of one possible pathway are discussed which may account for the greatly enhanced effectiveness of liposomal chemotherapy for experimental visceral leishmaniasis.

Leishmania braziliensis: Development of primary and satellite lesions in the experimentally infected owl monkey, Aotus trivirgatus

Twelve male and 8 female feral owl monkeys, Aotus trivirgatus, were inoculated intradermally at the dorsal base of the tail with 2 X 10(7) promastigotes (strains WR 128 or WR 539) or 5 X 10(5) amastigotes (strain WR 128) of Leishmania braziliensis panamensis, and the progression and regression of subsequent lesions were examined for up to 13 or 54 weeks after inoculation. Three of these monkeys had been infected previously with L. donovani, had been treated with meglumine antimoniate, and had recovered clinically from visceral leishmaniasis. All monkeys developed a cutaneous nodule at the inoculation site, but the size of the nodule varied (maximum 78 to 326 mm2 between 4 and 16 weeks after inoculation.) The initial nodule became ulcerated after 4 to 8 weeks in 17 of the 20 monkeys, and the ulcers persisted for 4 to 16 weeks until covered by a crust. Primary lesions disappeared by 17 to 52 weeks after inoculation, but satellite lesions, of similar morphology to the primary lesions but smaller, developed after 4 to 21 weeks in 14 of the monkeys. The primary nodule was excised in 4 monkeys at 6 weeks and did not recur nor did satellite lesions subsequently develop. The satellite lesions (median total number of 4, range 1 to 25) were adjacent to or at a maximum distance of 6 cm from the primary lesion, varied in size from 3 to 117 mm2, and persisted for 10 to 37 weeks. At 6 and 8 weeks after inoculation, tissue from the cutaneous leishmanial lesions from five monkeys was excised and examined. The granulomatous leishmanial lesions, located primarily in the dermis and subcutis, consisted of macrophages containing parasites, lymphocytes, plasma cells, and occasionally eosinophils. Satellite lesions at 14 weeks after inoculation were similar grossly and microscopically to the initial nodule. No significant differences were observed between promastigote or amastigote derived infections, between the two strains of L. b. panamensis, or between the course of infection based on the sex, age, karyotype, or country of origin of the owl monkeys. Cutaneous lesions developed when 5 X 10(5) amastigotes of L. b. panamensis (strain WR 128) were inoculated intradermally into the dorsal base of the tail, the upper eyelid, and the thorax of three monkeys. Leishmanial nodules which developed on the thorax regressed rapidly (after 2 to 5 weeks) whereas those on the upper eyelid and at the dorsal base of the tail persisted for 5 to 45 weeks after inoculation.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunization of mice with irradiated Trypanosoma cruzi grown in cell culture: Relation of numbers of parasites, immunizing injections, and route of immunization to resistance

Abstract Mice were given 5 or 8 weekly injectins of either 2·0 × 10 6 or 20·0 × 10 6 irradiated T. cruzi from cell culture (ratio of trypomastigotes to amastigotes, 1 : 1) via the intraperitoneal route or via the subcutaneous route and challenged via the subcutaneous route one week after the last injection with 5·0 × 10 4 T. cruzi in mouse blood. The irradiated parasites used were not capable of producing infections in either Vero cell cultures or C 3 H mice. Mice receiving irradiated parasites were significantly protected against the challenge infection as evidenced by significantly lower mean parasitemia, lessened signs of acute disease, and reduced mortality than that observed in untreated controls. Mice receiving 5 weekly immunizing injections of irradiated parasites were more resistant to challenge than those receiving 3 in previous work. Mice receiving 8 weekly immunizing injections were not significantly more protected against challenge than those receiving 5. Mice given 5 weekly injections of 20·0 × 10 6 irradiated parasites were significantly more resistant to challenge than those receiving 2·0 × 10 6 irradiated parasites on the same schedule. Mice given 5 weekly intraperitoneal injections of 20·0 × 10 6 irradiated parasites were significantly more resistant to challenge than those receiving an equivalent number of immunizing injections via the subcutaneous route.