Analysis of infection characteristics and antiparasite immune responses in resistant compared with susceptible hosts.
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Animals↗
Biomedical subjects
E Handman
Publications and source records attributed to E Handman.
Stage-specific, strain-specific, and cross-reactive antigens of Leishmania species identified by monoclonal antibodies.
The fusion of NS-1 myeloma cells with spleen cells from mice chronically infected with Leishmania tropica resulted in nine clones of hybridomas producing monospecific antibodies to membrane antigens of L. tropica. One of the antibodies (L-5-85) bound specifically to the promastigote form of the parasite, and the remaining eight recognized antigens shared by the promastigote and amastigote of L. tropica. Four of the antibodies (L-5-16, L-5-34, L-5-44, and L-2-3F11) detected parasite antigens on the surface of L. tropica-infected macrophages. Common antigens shared by L. tropica, L. mexicana, and L. donovani were identified as well as one antigen apparent on most Leishmania spp. and present also in Crithidia fasciculata. Two monoclonal antibodies (L-5-27 and L-5-41) were found to bind only to strains of L. tropica from simple cutaneous leishmaniasis. A special property shared by these two antibodies was the inhibition of parasite growth in macrophages in vitro.
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Resistance and abrogation of resistance to cutaneous leishmaniasis in reconstituted BALB/c nude mice.
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Animals↗
Resistance to cutaneous leishmaniasis in genetically susceptible BALB/c mice.
When injected cutaneously with promastigotes of an isolate of Leishmania tropica, BALB/c mice develop progressive cutaneous disease whereas lesions in BALB/c.H-2k mice heal after several weeks. Lesions in BALB/c mice injected into deep subcutaneous tissues with promastigotes are less obvious early but much more prominent later than in mice after strict intradermal injection. BALB/c mice injected with Corynebacterium parvum together with a preparation of frozen and thawed infected macrophages are more resistant to cutaneous disease than mice injected with either adjuvant or crude antigen mixture alone. Results of these experiments, and those on other mouse strains reported previously, will aid in the choice of mouse and injection regime to be used in testing the efficacy of isolated L. tropica antigens as vaccines.
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Identification and characterization of protein antigens of Leishmania tropica isolates.
Promastigotes of 4 Leishmania tropica isolates were biosynthetically labeled with 35S-methionine or surface radioiodinated, and the detergent lysates were analyzed by 2 dimensional gel electrophoresis. The protein patterns of cytoplasmic and membrane proteins detected in two independent isolates from simple cutaneous leishmaniasis cases were similar to each other, but were different from 2 isolates taken over a 20-yr interval from a case of leishmaniasis recidiva. The analysis of radioiodinated membrane proteins of L. tropica promastigotes revealed a simple pattern of 6 to 7 labeled major proteins and some minor ones. Unlike cytoplasmic proteins, membrane proteins were highly resistant to digestion by proteases. Partial cleavage of radioiodinated surface proteins by staphylococcal V-8 protease in the presence of 4.5 M urea and peptide mapping confirmed the presence of shared and isolate-specific proteins. The significance of these findings for the classification of Leishmania spp. and the pathogenesis of the different disease states that they cause are discussed.
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Cutaneous leishmaniasis in mice: disease patterns in reconstituted nude mice of several genotypes infected with Leishmania tropica.
BALB/c mice differ from CBA/H, (CBA/H x BALB/c)F1 and C57BL/6 mice in being highly susceptible to infection with a particular isolate of the intramacrophage protozoan parasite, Leishmania tropica. This is true over a wide range of challenge doses of promastigotes administered intradermally although, at low doses, male BALB/c are able to restrict the rate at which cutaneous lesions increase in size. Resistance to infection in highly susceptible hypothymic nude (nu/nu) mice of CBA/H and C57BL/6 genotypes can be achieved readily with as few as 10(6) syngeneic lymphoid cells. In CBA/H.nu/nu mice, Lyl+2- cells in low dose cellular inocula appear to be involved in restoring resistance (i.e. rapid resolution of infection). Surprisingly, a proportion of BALB/c.nu/nu mice injected with syngeneic lymphoid cells are resistant and thus differ from intact BALB/c mice or non-reconstituted BALB/c.nu/nu mice. In keeping with a high efficacy of limited numbers of T cells following minimal reconstitution, the majority of nude mice implanted subcutaneously with allogeneic, irradiated, neonatal thymus grafts are resistant to L. tropica infection. Genetic features of the nude mouse, such as defect in skin, may contribute to the extraordinary potency of T cell reconstitutive manipulations. Evidence was obtained that BALB/c.nu/nu skin on BALB/c.nu/+ recipients did not support the development of lesions as readily as normal skin sites.
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Antibodies to Leishmania tropica promastigotes during infection in mice of various genotypes.
After intradermal injection of 10(7) promastigotes of a particular isolate of the intramacrophage protozoan parasite, Leishmania tropica, the development of disease (cutaneous lesions) is much more severe in BALB/c than in three other mouse strains, C57BL/6,C3H/He and CBA/H. Using fixed promastigotes and 125I-labelled protein A in a solid-phase radioimmunoassay (RIA), titres of antibody were shown to increase up to about day 50 of infection. However, titres were not markedly different in BALB/c mice compared with the other three resistant strains, although antibody levels were highest in sera from the diseased BALB/c mice at late time points. Using isotype-specific antisera in the RIA, and sera from the day 50 time point, the isotype distribution of anti-promastigote antibodies was not noticeably different in sera from mice of the four genotypes with IgG1 and IgG2a (+/- IgG2b) antibodies predominating. It is concluded that differences in susceptibility to disease in this murine model of cutaneous leishmaniasis do not correlate with any decrease or increase in any particular antibody response to the promastigote.
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Binding of lectins to the cell surface of Trypanosoma cruzi.
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Animals↗
Delayed hypersensitivity to Toxoplasma and unrelated antigens in Toxoplasma-infected mice: induction and elicitation of delayed-type hypersensitivity by antigen-pulsed macrophages.
Delayed-type hypersensitivity (DTH) to Toxoplasma and unrelated antigens in Toxoplasma-infected BALB/c mice was investigated by the radioisotopic uptake method of Vadas et al. (Int. Arch. Allergy Appl. Immunol. 49: 670-692, 1975). DTH became positive on day 30 of infection and remained positive during chronic infection. The expression of DTH in mice infected with the relatively avirulent C37 strain of the parasite paralleled the Toxoplasma antibody response as detected by the Sabin-Feldman dye test. Mice sensitized with Toxoplasma, keyhole limpet hemocyanin, or sheep erythrocytes during the acute or chronic phase of Toxoplasma infection showed a DTH reaction similar to that of uninfected sensitized controls. No parasite antigens could be detected by immunofluorescence techniques on the surface of Toxoplasma-infected cells. When killed organisms were added to the cell cultures, specks of fluorescence appeared on cells containing intracellular parasites as well as on cells without intracellular organisms. That the antigens may be present in or on macrophages in a form readily recognizable by T cells is suggested by experiments in which we demonstrated that injection of uninfected normal macrophages pulsed with Toxoplasma-soluble antigens into the ears of chronically infected mice elicited a DTH reaction comparable to that observed when 10(6) Formalin-fixed tachyzoites were used as the test antigen. When macrophages pulsed with Toxoplasma antigen were used in attempts to induce DTH in naive uninfected mice, the intensity of the reaction was similar to that observed in infected mice.
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Antibody responses to toxoplasma antigens in mice infected with strains of different virulence.
Swiss Webster mice were infected with either the relatively virulent C56 strain or the relatively avirulent C37 strain of Toxoplasma gondii, and the sequence of their antibody response to surface antigens of the parasite was studied. An immunoglobulin M (IgM) agglutinating antibody was the first serologically detectable antibody and was first detected on day 2 in mice infected with the C37 strain and on day 5 in mice infected with the C56 strain. IgG antibodies were first detected on day 8 for both strains. The major component of the IgG antibodies was IgG2:IgG3 antibodies had lower titers, and no IgG1 antibodies were detected. The IgG antibodies were active in direct parasite agglutination and in the complement-dependent cytotoxicity assay of Sabin and Feldman (Science 108:660-663, 1948). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, sera from mice infected with T. gondii detected all major radioiodinated surface proteins of toxoplasma tachyzoites. The earliest time point at which these antigens were detected differed for the two strains. Serum from mice infected with C56 strain immunoprecipitated all cell surface antigens by day 10 of infection, whereas serum from mice infected with the C37 strain did not do so until day 15 of infection.
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Use of monoclonal antibodies to detect antigens of Toxoplasma gondii in serum and other body fluids.
Monoclonal antibodies to Toxoplasma gondii were used in an enzyme-linked immunosorbent assay to detect antigens of the parasite in toxoplasma lysate, in peritoneal fluid of mice, and in sera from humans acutely infected with T. gondii. Four of the six monoclonal antibodies were able to detect antigens of toxoplasma in these specimens. Control sera from individuals not infected with T. gondii and from individuals chronically infected with the parasite were negative in the enzyme-linked immunosorbent assay. Sera from individuals not infected with T. gondii but with positive titers from rheumatoid factor were also negative; 2 or 10 sera from individuals not infected with T. gondii but with positive titers for antinuclear antibodies reacted with the monoclonal antibodies. When the results of the enzyme-linked immunosorbent assay with monoclonal antibodies and with the F(ab)2 fraction of an immunoglobulin G from a rabbit infected with T. gondii were compared, it was noted that the F(ab)2 was more active in detecting parasite antigens than were the monoclonal antibodies. Thus, although monoclonal antibodies can be used to detect antigens of T. gondii in sera and other body fluids, polyvalent antibody (such as the F(ab)2 fraction) appears to be more satisfactory for this purpose.
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Serological and immunochemical characterization of monoclonal antibodies to Toxoplasma gondii.
The fusion of mouse NS-1 myeloma cells with spleen cells from mice chronically infected with Toxoplasma gondii resulted in eight clones of hybridomas producing monospecific antibodies against membrane or cytoplasmic antigens of Toxoplasma tachyzoites. One of the antibodies to a cytoplasmic determinant was an IgM; the others directed to membrane or cytoplasmic antigens belonged to the IgG2 or IgG3 isotypes. Antibodies of clones 1E11 (IgG3), 2G11, and 3E6 (IgG2) directed to membrane antigens, bound complement and were reactive in the complement-dependent cytotoxicity assay of Sabin-Feldman. These IgG2 antibodies were strongly agglutinating to parasites, whereas the IgG3 was relatively weak. Another IgG2 antibody (5B6), possibly recognizing a shared antigen of membrane and cytoplasm, exhibited a low titre in the cytotoxicity assay as well as in the agglutination assay. Two other antibodies to membrane antigens (2B7 and 2F8) as well as an antibody to a cytoplasmic antigen (3G3) did not bind complement and did not cause agglutination. The pattern of parasite staining produced by monoclonal antibodies to membrane antigens in an IFA test was different from that of polyvalent antisera. A strictly localized or 'beaded' staining was observed, as well as a smooth, rim fluorescence. Toxoplasma tachyzoites were surface radio-iodinated and the solubilized membrane proteins were immunoprecipitated with monoclonal antibodies and analysed by two-dimensional polyacrylamide gel electrophoresis. Two independently arising monoclonal antibodies to membrane antigens (2G11 and 3E6) consistently precipitated both the solubilized 35,000 and 14,000 mol. wt proteins, while 1E11 precipitated the 27,000 mol. wt protein.
Agglutination Tests↗
Detection and characterization of membrane antigens of Toxoplasma gondii.
Toxoplasma gondii tachyzoites were surface radioiodinated by the lactoperoxidase technique, and the solubilized membrane proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis. Four major labeled proteins with apparent m.w. of 43,000, 35,000, 27,000, and 14,000 were detected. None of the radioiodinated proteins bound to concanavalin A-Sepharose. When a panel of eight different fluorescein-conjugated lectins was used in an attempt to characterize further the nature of the cell membrane, none of the lectins bound to intact tachyzoites. Two-dimensional polyacrylamide gel electrophoresis did not reveal any significant differences among three different strains of Toxoplasma. Each of the radioiodinated surface proteins was precipitable by sera from mice chronically infected with the same strain as well as by a series of sera from mice infected with other strains. Sera from humans with acute Toxoplasma infection showed more variability in that some precipitated all labeled proteins whereas others precipitated only two or three of them. Monoclonal antibodies (2G11 and 3E6) prepared by hybridization of spleen cells from Toxoplasma-immune mice with myeloma cells consistently precipitated both the solubilized 35,000 and 14,000 dalton proteins, whereas 1E3 precipitated the 43,000-dalton protein and 1E11 the 27,000-dalton protein.
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Murine cutaneous leishmaniasis: disease patterns in intact and nude mice of various genotypes and examination of some differences between normal and infected macrophages.
The course of the disease, cutaneous leishmaniasis, caused by the intracellular protozoan parasite Leishmania tropica, differs markedly amongst various common inbred mouse strains. After intradermal injection of 1 x 10(6) promastigotes to young female specific pathogen-free (SPF) derived mice, persistent infection characterized by an expanding ulcerous lesion is seen in BALB/c and DBA/2 mice. In the strains CBA/H, C3H/He and A/J, lesions resolve within 8 weeks, and in C57B1/6 mice no real lesion typical of cutaneous leishmaniasis develops at the injection site. NZB mice are highly resistant. Macrophages harvested from the thioglycollate-stimulated peritoneal cavity of NZB and C57B1/6 mice appear to differ from macrophages of the other mouse strains in not supporting multiplication of L. tropica organisms in vitro. Nevertheless, hypothymic nude (nu/nu) mice of C57B1/6 genotype, as well as CBA/H-nu/nu and BALB/c-nu/nu mice, develop large lesions with metastases to other cutaneous and visceral locations. In the intact mice in which infection resolves spontaneously, resistance to reinfection is complete. Using mouse antipromastigote sera and an indirect fluorescent antibody test in carefully controlled experiments, L. tropica antigens were detected on in vitro infected macrophages of both highly susceptible BALB/c and relatively resistant CBA/H genotypes. After incubation with a crude soluble antigen preparation from cultured promastigotes, infected BALB/c macrophages differed from infected CBA/H macrophages (and uninfected macrophages of both genotypes) in being unable to sensitize syngeneic recipients for a delayed-type hypersensitivity response to that antigen. When infected and uninfected macrophages were used as "blocking cells" in an in vitro alloreactive cytotoxic T cell system involving cells from congenic mice, evidence was obtained for reduced H-2d expression on infected macrophages of the susceptible mouse strains, BALB/c. The data in this model system of cutaneous leishmaniasis raise the possibility that genetic susceptibility is associated with both a permissive macrophage and defective T cell recognition of parasite antigens on infected macrophages. Defective recognition may be the result of reduced functional expression of H-2d antigens on infected BALB/c macrophages required for efficient recognition by syngeneic T cells of one or more subpopulations.
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Stimulation by granulocyte-macrophage colony-stimulating factor of Leishmania tropica killing by macrophages.
The intracellular protozoan parasite Leishmania tropica was found to survive unharmed and to multiply for several days in normal mouse peritoneal macrophages. In contrast, when infected monolayers were treated with GM-CSF, there was a continuous decrease in the percentage of infected cells, reaching less than 10% on day 4 in culture, compared to about 30% in normal controls. Microscopic observations showed an increased number of dead parasites in GM-CSF treated infected cells. Within 5 hr of incubation with GM-CSF, almost 40% of intracellular parasites showed morphologic damage, compared to less than 10% in untreated cells. Pretreatment of macrophage monolayers with pure GM-CSF before infection led to an increased level of phagocytosis of L. tropica parasites as reflected by the percentage of infected cells and the increased number of parasites in each infected cell. GM-CSF treated cultures showed 73% infected cells containing a mean of five parasites per cell, as compared to controls in which only about 50% of macrophages were infected with only two parasites per cell. The number of dead parasites per cell was 5-fold higher in the GM-CSF treated cultures at 2 hr. After 24 hr the percentage of infected GM-CSF treated cells was less than one-third that in the control cultures.
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Protection of mice against plasmodium and babesia infections: attempts to raise host-protective sera.
In an attempt to generate large numbers of mice resistant to Plasmodium berghei and Babesia rodhaini to be used as donors of antibody-secreting cells for hybridoma production, various methods of inducing resistance to repeated challenge with infected blood cells have been explored. Although results of independent experiments varied markedly, prior injection of CBA/M mice with BCG, and prior infection of BALB/c mice with Plasmodium yoelii, were found to be manipulations capable of inducing resistance to P. berghei. A single dose of serum, harvested from resistant mice challenged several times with P. berghei, could transfer resistance against P. berghei to a proportion of naive CBA/H recipients. Although resistance to multiple B. rodhaini challenge could be induced in mice, in no situation was a host protective effect of a single high dose of serum demonstrated in naive recipients.
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Observations on immunization against cutaneous leishmaniasis in Israel.
Trials of immunization against Leishmania tropica were initiated in the Israel Defense Forces in 1968. The rate of takes has declined gradually over the years. In 1975, 425 soldiers were inoculated with the same strain of L. tropica as that used since 1968; they were examined at three- and six-month intervals after the inoculation, with 291 soldiers undergoing at least one follow-up examination. Only 13.7% developed lesions at the inoculation site, usually between three and six months after the inoculation. Leishmanin tests were performed in 220 soldiers and there were positive reactions in 19.5%. There was a fair correlation between the development of clinical lesions and positive leishmanin tests. A positive leishmanin test, or a typical lesion, or both, was seen in 21.3% of the inoculated soldiers. L. tropica tends to lose its virulence after prolonged storage and multiple passages. A new strain isolated a few months before this trial was used to inoculate 50 soldiers, 31 of whom were followed up. All 31 developed typical lesions at the site of inoculation, most of them within two to four weeks following the inoculation.
Follow-Up Studies↗
Promotion of leishmanial infections in non-permissive host macrophages by conditioned medium.
Leishmania enriettii grows well in guinea pig macrophages in culture but fails to do so in mouse macrophages. Dialyzed and concentrated conditioned medium from L. enriettii cultures promoted the infection in the mouse macrophages at 37 degrees C and at 32 degrees C. Similarly processed medium on which L. tropica had been cultured did not cause the same effect. Immune precipitation of the conditioned medium by anti-Leishmania serum cancelled out the effect. Infections of guinea pig macrophages by L. tropica were also enhanced by the homologous medium, but we were unable to quantitate the effect.
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