Profile of human T cell response to leishmanial antigens. Analysis by immunoblotting.

Control and resolution of leishmanial infection depends primarily on T cell-mediated immune mechanisms. The nature of the leishmanial antigens involved in eliciting T cell immunity is unknown. We have examined the pattern of peripheral blood lymphocyte responses in patients with active, healed, or subclinical leishmanial infection to fractionated leishmanial antigens using a T cell immunoblotting method in which nitrocellulose-bound leishmanial antigen, resolved by one or two dimensional electrophoresis, are incorporated into lymphocyte cultures. The proliferative and IFN-gamma responses of cells from patients with healed mucosal or cutaneous leishmaniasis were remarkably heterogeneous and occurred to as many as 50-70 distinct antigens. In contrast, responses from subjects with active, nonhealing, diffuse cutaneous leishmaniasis were either absent or present to only a small number of antigens. Control and resolution of leishmaniasis, and resistance to reinfection, is therefore associated with a T cell response to a large and diverse pool of parasite antigens. The method of T cell immunoblotting appears to offer a powerful, rapid, and relatively simple approach to the identification of antigens involved in eliciting a T cell response in human leishmaniasis.

Relationship of serum complement levels to events of the malarial paroxysm.

Malarial paroxysms due to Plasmodium vivax were studied for alterations in whole serum complement (C') and certain C' components. The objective was to relate C' values with events of the parasite cycle during schizogony and with the febrile pattern. Substantial decreases in C' were found in 9 of 18 paroxysms studied during relapse. In contrast, only one of 22 paroxysms occuring during the primary attack was associated with a striking depression in C', and this case exhibited certain characteristics of a relapse paroxysm. The mean change in C' levels during paroxysms in relapse (-23%) was significantly different from paroxysms of the primary attack (-2%). Depletion of C' was associated directly with degree of parasitemia and presence of complement-fixing (CF) antibody. Lowest levels of C' were found within a few hours after completion of schizont repture and peak fever. C4 levels reflected changes in whole serum C' and appeared to be a more sensitive indicator of C' alterations during malaria. While the alterations in C4 as well as C1 and C2 indicated that the classical C' pathway was involved, some preliminary results showed little or no depletion of late components, C3 and C6. Overall results are compatible with C' activation and depletion during or soon after schizont repture if parasite density is sufficiently high and if CF antibody is present.

In vivo cytokine profiles in patients with kala-azar. Marked elevation of both interleukin-10 and interferon-gamma.

The immunological mechanisms underlying the susceptibility to disseminated visceral parasitism of mononuclear phagocytes in patients with kala-azar remain undefined. Resistance and susceptibility are correlated with distinct patterns of cytokine production in murine models of disseminated leishmanial disease. To assess lesional cytokine profiles in patients with kala-azar, bone marrow aspirates were analyzed using a quantitative reverse transcriptase PCR technique to amplify specific mRNA sequences of multiple Th1-, Th2-, and/or macrophage-associated cytokines. Transcript levels of IL-10 as well as IFN-gamma were significantly elevated in patients with active visceral leishmaniasis; IL-10 levels decreased markedly with resolution of disease. These findings suggest that IL-10, a potent, pleiotropic suppressor of all known microbicidal effector functions of macrophages, may contribute to the pathogenesis of kala-azar by inhibiting the cytokine-mediated activation of host macrophages that is necessary for the control of leishmanial infection.

Strongyloides stercoralis hyperinfection in a carrier of HTLV-I virus with evidence of selective immunosuppression.

A patient with near fatal Strongyloides hyperinfection syndrome is briefly described. Investigation for possible risk factors for this parasitic infection disclosed that he was a carrier of human T-cell leukemia virus type I (HTLV-I), but without evidence of disease due to this retrovirus. Over the next few years, the patient's serum antibody levels of IgG to S. stercoralis larvae declined and became undetectable despite continued infection with the parasite. Repeated courses of appropriate treatment cleared the parasitic infection only temporarily. The patient was also found to have undetectable total serum IgE and a negative immediate hypersensitivity skin test to S. stercoralis antigens. Five of six other patients with HTLV-I-associated disease and with or without strongyloidiasis were also found to have very low total serum IgE levels. It is postulated that HTLV-I infection in certain individuals may selectively impair immune responses that are critical in controlling strongyloidiasis.

Antigens from the surface and excretions/secretions of the filariform larva of Strongyloides stercoralis.

The surface and excretory/secretory (ES) antigens of the infective, filariform larva (L3) of Strongyloides stercoralis were identified. These studies provide a basis for the purification of these proteins as diagnostic allergens for human strongyloidiasis. The Mr values of the surface and ES molecules were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, fluorography, or silver staining following the recovery of these molecules after the radiolabelling of living parasites. At least 10 surface proteins were radioiodinated extrinsically using chloroglycoluril as the catalyst for iodination, and then extracted with detergents and/or beta-mercaptoethanol. Several surface molecules of the L3 were immunogenic in humans, as determined by immunoprecipitation with sera (IHS) from infected patients. About 30 proteins were present in the ES preparation. Many ES antigens were labelled biosynthetically during the culture of larvae in media supplemented with either [35S]methionine or [14C]glucose. Furthermore, several of the surface proteins of the L3 were found with the ES antigens recoverable by culturing larvae in vitro. About 10 of the ES proteins were immunogenic as determined by immunoaffinity chromatography using IHS; and two of these antigens with Mr 50,000 and 90,000 incorporated [35S]methionine during culture of larvae. Moreover, some ES proteins were allergenic when tested in an in vitro assay of histamine release from basophils from infected humans or monkeys. The isotype of the homocytophilic antibodies involved in this immediate hypersensitivity assay, which is the basis of a diagnostic skin test for human strongyloidiasis, appears to be IgE.

Leishmaniasis affecting the eyelids.

Leishman-Donovan bodies were recognized in the smear of a biopsy specimen from an eyelid ulcer. The infecting organisms were identified serologically as Leishmania braziliensis panamensis. The ulcer responded to pentavalent antimony. Ultrastructurally, the organisms had double-unit membranes, beneath which lay a palisade of microtubules. At one end of the organism, there was a rudimentary flagellum; at the other, the nucleus. A kinetoplast basal complex separated the two.

Effect of temperature on multiplication of Leishmania amastigotes within human monocyte-derived macrophages in vitro.

Leishmania tropica, a cause of cutaneous leishmaniasis, multiplied more rapidly within human macrophages in vitro at 35 degrees C than at 37 degree C, and was almost completely eliminated at 39 degrees C. In contrast, Leishmania donovani, the cause of the visceral leishmaniasis, multiplied equally well at 35 degrees C and at 37 degrees C,, and was only 40% eliminated at 39 degrees C. This in vitro study suggests that the localization of the two strains to cooler and warmer regions of the body, respectively, is at least partially explained by the inherent temperature sensitivity of the parasite-macrophage unit. The striking elimination of this strain of L. tropica within macrophages at 39 degrees C may make this model suitable for predicting the clinical response of cutaneous strains to heat therapy.

A complement-fixing antigen from Trypanosoma cruzi grown in cell cultures.

A complement-fixing (CF) antigen was prepared from amastigotes and trypomastigotes of Trypanosoma cruzi (Ernestina strain) grown in beef embryo cell cultures. Multiple lots of the antigen, which consisted of a supernate of washed and disrupted organisms, required material from 10(6) to 10(7) total organism per ml for optimum CF activity. Antibody at dilutions up to 1:256 was demonstrable in various sera from infected animals or patients. Contaminating beef cells from infected cultures were shown to be partly responsible for crossreactions of the antigen by CF with sera from cases of cutaneous leishmaniasis in whom concomitant infection with T. cruzi could be excluded. There were no cross-reactions with syphilitic sera and the frequency of positive reactions with normal sera was very low. Some characterisitics of the antigen included stability to storage at -20 degrees C and -70 degrees C for months, inactivation at 60 degrees C and by lyophilization, and an estimated molecular size of between 50,000 and 100,000 on the basis of membrane filtration.

Comparison of cell culture with epimastigote antigens of Trypanosoma cruzi.

Various epimastigote antigens of Trypanosoma cruzi were compared with an amastigote-trypomastigote antigen from infected cell cultures by complement fixation (CF) and gel diffusion (Ouchterlony). CF results with human Chagasic sera showed that the amastigote-trypomastigote antigen was usually more sensitive than epimastigote antigens tested. In addition, cross-reactions with normal and other sera were no greater and perhaps less frequent than with crude epimastigote antigens. Specificity of the amastigote-trypomastigote antigen, however, was less than with a protein extract of epimastigotes. Gel diffusion results with human Chagasic and hyper-immunized rabbit sera indicated differences between epimastigote and amastigote-trypomastigote antigens whereas differences by CF with the same sera were equivocal.

Thermosensitivity patterns of Old vs. New World cutaneous strains of Leishmania growing within mouse peritoneal macrophages in vitro.

Infection of mouse peritoneal macrophages in vitro was used to examine the effect of elevated temperature on the intracellular replication of various strains of Leishmania. Of eight cutaneous strains examined, all grew optimally at 35 degrees C. At 37 degrees C the reduction in growth was most pronounced for the New World cutaneous strains, and at 39 degrees C three of four New World cutaneous strains were completely destroyed whereas all of the L. tropica strains survived and exhibited at least 100% growth after 3 days. The results of these in vitro studies correlate closely with the outcome of heat therapy on two patients with cutaneous disease, suggesting that, in general, cutaneous lesions due to L. tropica strains might be less responsive to heat therapy than lesions due to L. mexicana and related strains.

Cutaneous leishmaniasis--a case with persistent organisms after treatment in presence of normal immune response.

A Peace Corps volunteer in Senegal, West Africa contracted cutaneous leishmaniasis which had several noteworthy features. One of the three presenting cutaneous ulcers was associated with subcutaneous nodules and viable organisms were recovered from healing lesions after multiple courses of treatment, including amphotericin B. Yet, the patient was found to exhibit both humoral and cell mediated features of normal immunologic responsiveness. Ultimately, clinical and parasitological cure occurred. The patient's organism was found to produce lesions in the foot pads of mice.

Molecular differences between several species of Strongyloides and comparison of selected isolates of S. stercoralis using a polymerase chain reaction-linked restriction fragment length polymorphism approach.

The relationships between the parasitic nematodes of medical importance belonging to the genus Strongyloides was studied using a polymerase chain reaction (PCR)-linked restriction fragment length polymorphism approach. We used several human and dog isolates of S. stercoralis, a monkey isolate of S. fulleborni, and S. ratti, a rodent parasite. The molecular analysis was based on amplification of the internal transcribed spacer and the 5' portion of the 23S-like rRNA gene followed by restriction enzyme digests. The length of the PCR product was specific to each species and varied around 1.5 kilobase pairs. Using nine restriction enzymes, we were able to analyze both interspecific and intraspecific variations. With four restriction enzymes (Taq I, Dde I, Dra I, and Mwo I), human isolates of S. stercoralis from different parts of the world showed identical patterns and could be differentiated from the dog isolate of S. stercoralis. Interspecific differences were readily observed with these and other enzymes. In addition to providing new information on the genomic characteristics of Strongyloides parasites, the results suggest that this technique could be useful for diagnostic and epidemiologic investigations.

Comparative sensitivity and specificity of ELISA and IHA for serodiagnosis of strongyloidiasis with larval antigens.

Sera from 68 patients with parasitologically proven strongyloidiasis were tested by the ELISA and IHA tests using larval antigens prepared from Strongyloides stercoralis and Strongyloides ratti. The ELISA using the S. stercoralis antigen detected the greatest number of sero-reactors (83.8%), whereas the IHA using the S. ratti antigen detected the fewest (55.9%). In addition, the S. stercoralis antigen had higher geometric mean titers than the S. ratti antigen in both the ELISA and the IHA tests. Sera from 37 presumed normal individuals also were tested by IHA and ELISA and nonspecific reactions were seen only with the IHA test. When sera from patients with parasitic infections other than strongyloidiasis were tested, the only consistent cross-reactions were with those sera from patients who had occult filariasis and acute schistosomiasis.

Late metastatic Leishmaniasis in the mouse. A model for mucocutaneous disease.

BALB/c, C57B1/6 and (BALB/c x C57B1/6)F1 mice all proved susceptible to infection by a strain of Leishmania isolated from a Central Brazilian with espundia. The course of disease differed markedly between BALB/c and C57B1/6 mice. BALB/c mice suffered from a rapidly progressive and widely metastatic, but non-ulcerative, disease resembling diffuse cutaneous leishmaniasis. In contrast, C57B1/6 mice initially contained parasite multiplication effectively and appeared clinically cured. However, the parasite could persistently be cultured up to about 1 year post-infection. At that time, the parasite load in the infected footpad increased and a patent disease developed characterized by distinctive ulcerative metastases with destruction of soft-tissue in the nasal region similar to the one observed in espundia. Development of disease in both strains of mice was associated with depression of cell-mediated immunity as monitored by delayed-type hypersensitivity in vivo and lymphocyte transformation in vitro. Thus, our study suggests that diffuse cutaneous leishmaniasis and espundia can be caused by the same strain of parasite, and that the particular clinical expression in the individual mouse is determined by the host response.

Specific and non-specific lymphocyte blastogenic responses in individuals infected with Trypanosoma cruzi.

Lymphocyte blastogenic response to various parasite-specific and non-parasite antigens was measured in 31 Trypanosoma cruzi-infected individuals representing the indeterminate, megadisease and cardiomyopathy forms of Chagas' disease. The non-parasite antigens and mitogens stimulated responses in the peripheral blood mononuclear cell (PBMC) cultures of the infected individuals to levels not different than the stimulation seen in PBMC of the normal control group (n = 10). The cell culture antigen (CC-Ag) derived from trypomastigote-amastigote forms of T. cruzi and the epimastigote antigen (EPI-Ag) elicited a strong response among all infected individuals in contrast to virtually no response among the normal controls. Comparisons among the individuals with T. cruzi infection stimulated with EPI-Ag showed no significant difference between the clinical groups. However, stimulus with CC-Ag showed the megadisease group response to be greater than the response of the indeterminate group, but not different than than of the cardiomyopathy group. The possible biological implications of these findings are discussed.

Persistent strongyloidiasis in an immunodeficient patient.

A 56-year-old woman with acquired, common variable immunodeficiency was found to have persistent gastrointestinal as well as pulmonary infection with Strongyloides stercoralis. Repeated courses of treatment with thiabendazole led to marked reduction or loss of Strongyloides stercoralis larvae, but cessation of treatment always led to recurrence of Strongyloides infection. Several small bowel biopsies showed normal villous architecture and little inflammatory response to presence of larvae. Interestingly, no definite symptomatology could be attributed to the Strongyloides infection. It was postulated that the lack of signs and symptoms of strongyloidiasis, as well as poor response to treatment, was related to the immunodeficiency state. With low-dose, long-term interrupted courses of thiabendazole treatment, the Strongyloides infection finally seemed to be cured.

Skin test antigens for immediate hypersensitivity prepared from infective larvae of Strongyloides stercoralis.

More rapid and simplified diagnostic procedures are needed for the diagnosis of strongyloidiasis. One approach is the use of an immediate hypersensitivity skin test that would reliably identify infected people. Accordingly, somatic and excretion/secretion (E/S) antigens were prepared from filariform larvae of Strongyloides stercoralis and were treated to remove possible adventitious agents. By use of a quantitative method for measurement of skin reactions, several preparations of the 2 antigens were tested in uninfected controls and in various groups of patients. Doses of 0.35 microg of E/S and 4 microg of somatic antigens elicited positive skin tests in 82-100% of infected people, depending on clinical status. A lower frequency of positive skin tests was found in strongyloidiasis patients also infected with human T-cell lymphotropic virus type 1. Cross-reactions, especially to somatic antigens, were frequently found in patients with filarial infections. Despite these limitations and the need for further study of specificity, these results provide a basis for future development of a diagnostic skin test antigen for strongyloidiasis.

Observations on local heat treatment for cutaneous leishmaniasis.

Local heat treatment was tested and found effective in three patients with diffuse cutaneous leishmaniasis (DCL), a form of disease poorly responsive to the usual chemotherapy. A water bath that circulated water through a pad wrapped around the lesion provided a temperature of 39 degrees C to 41 degrees C for a cumulative time of at least 20 hours, over a period of several days. In the DCL patients beneficial effect of heat treatment was documented by pre- and post-treatment biopsies and cultures. Several other patients with ordinary cutaneous leishmaniasis did not respond to the same form of treatment. It was concluded that different strains and/or species of leishmanial parasites vary in their sensitivity to elevated temperature. While local heat treatment may be curative in certain cases of cutaneous leishmaniasis, such therapy is still experimental and should be monitored by quantitative parasitological studies to document its usefulness.

Experimental disseminated strongyloidiasis in Erythrocebus patas. I. Pathology.

Fatal disseminated disease was induced in ten patas monkeys infected with two Southeast Asian strains of Strongyloides stercoralis. While some animals died within 6 weeks after infection, others controlled their infections until placed on high doses of corticosteroids. Larvae were first noted in the stools 11-20 days after transcutaneous exposure to filariform larvae. Daily larval counts tended to increase as the infections progressed, but the number of larvae in the stool was not predictive of whether a monkey would control his infection or succumb to fatal disease. Hyperinfection was confirmed in the six monkeys in which counts were made of the adult female parasites in the duodenum at postmortem, as well as by pathologic findings in all animals. Clinical signs of disease were vague until dyspnea induced by terminal pulmonary hemorrhage occurred. Eosinophilia and/or basophilia were noted intermittently in some infections. Severe necrotizing duodenitis, colitis, and pulmonary hemorrhage were the most conspicuous postmortem findings. Hyperinfection has been predictably induced in a cercopithecoid monkey for the first time; a species which may lend itself to further investigations into the pathogenesis of disseminated strongyloidiasis .

Experimental disseminated strongyloidiasis in Erythrocebus patas. II. Immunology.

Parasite-specific humoral and cellular immune responses were evaluated in seven Erythrocebus patas monkeys experimentally infected with a Southeast Asian strain of Strongyloides stercoralis. Most animals developed high titers of anti-larval surface IgG antibody (as evaluated by the indirect immunofluorescence test), and all animals tested developed specific IgE antibody (as shown by the in vitro histamine release test). Modest lymphoproliferative responses to S. stercoralis antigens were demonstrated in most animals during the early phase of the infection (days 20-40), but disappeared later. Steroid treatment (prednisone, 12.5 mg/kg on alternate days) was given to three animals, but did not appear to significantly affect the immune parameters tested. The degree of the immune responses to S. stercoralis larval antigens did not correlate well with the course of the infection, and several animals died of disseminated disease in spite of demonstrable humoral and cellular responses to these antigens. We suggest therefore that other factors, such as local intestinal immune and nonimmune mechanisms may be of importance in protection against disseminated strongyloidiasis.