Dendritic epidermal gamma/delta T cells (DETC) activated in vivo proliferate in vitro in response to Mycobacterium leprae antigens.

BACKGROUND: gamma/delta T-cell receptor (TCR)+ dendritic epidermal T cells (DETC) are part of a primitive defense system in the skin; they are capable of responding only to a limited number of antigens. The aim of the present study was to test whether DETC can proliferate in vitro in response to antigens of Mycobacterium leprae. METHODS: DETC were obtained from CBA mouse ear skin by trypsinization and Histopaque gradient centrifugation. The resulting epidermal cell suspension contained up to 20% DETC, as analyzed by the fluorescence activated cell sorter (FACS) after staining with anti-Thy-1 or anti-gamma/delta TCR monoclonal antibodies (mAbs). The freshly isolated cells, or DETC cultured up to 4 weeks with interleukin-2 (IL-2), were exposed in vitro for up to 6 days to varying doses of the following M. leprae antigens: (1) integral (live) M. leprae bacilli; (2) Dharmendra antigen; and (3) PGL-1 (phenolic glycolipid of M. leprae). The DETC response was assessed by tritiated thymidine (3H-TdR) incorporation. RESULTS: The freshly isolated DETC, or DETC cultured up to 4 weeks with IL-2, did not respond significantly to any of the M. leprae antigens, although at the same time they were able to respond vigorously to concanavalin A (Con A), as positive control. If, however, DETC were isolated from skin, painted 7 days before with croton oil (10 microL/cm2 to cause irritant dermatitis, they were able to respond to all M. leprae antigens by a 3-4-fold incrase in the 3H-TdR uptake. The most effective stimulator was a 1 : 1 mixture of Dharmendra and PGL-1 (0. 01 microg/mL), which was as effective as 10-fold higher doses of either antigen alone. Cell counts confirmed that increased DNA synthesis was associated with cell proliferation. Experiments employing alpha/beta-TCR CBA murine spleen cells and epidermal cell suspension treated with anti-gamma/delta or antialpha/beta mAbs + C' proved that only the gamma/delta DETC were the responder cells to M. leprae antigens. CONCLUSIONS: The results suggest that activation of DETC in vivo may make them responsive to M. leprae antigens. A significant increase in the number of class II major histocompatibility complex (MHC) positive, nondendritic cells was observed in the croton oil-treated epidermis. We hypothesize that croson oil-induced upregulation of class II MHC expression, which endows epidermal cells with antigen-presenting capabilities, might be an important factor in vivo in delivering an immunogenic signal to resident DETC in the skin.

Lepromin responses in recipients of a candidate antileprosy bacterin vaccine (WHO-IMMLEP Mycobacterium leprae killed preparation) in the USA.

BACKGROUND: Identification of the nine-banded armadillo as a potential source of massive numbers of Mycobacterium leprae led to the development of a candidate bacterin vaccine for possible immunoprophylaxis. METHODS: Volunteers were from a leprosy-hypoendemic, nonBCG-using area (USA). They had been vaccinated intradermally 3 years earlier with a candidate antileprosy bacterin vaccine of irradiated and autoclaved Mycobacterium leprae obtained from experimental nine-banded armadillos. They were tested for dermal responsiveness to standard lepromin A. RESULTS: Values for induration and erythema appeared slightly greater for the vaccinated group; however, the differences were not statistically significant, indicating no appreciable 'anamnestic' effect on either Fernandez (early) or Mitsuda (late) reactions after 3 years. CONCLUSIONS: Because previous studies had demonstrated that administration of this bacterin produced no humoral changes, it now appears less probable that laboratory methods will be of much help in assessing even possible effectiveness of such vaccination.

Lepromin skin testing in the classification of Hansen's disease in the United States.

Hansen's disease, or leprosy, although a relatively uncommon disease in the United States, continues to be important because of its implications--physical, psychological, and social--for the patient. Prognosis and treatment of the disease are based largely on clinical classification, which ranges from the multibacillary "lepromatous" to the paucibacillary "tuberculoid" forms, depending on the patient's specific immune capabilities. Traditionally, skin testing with lepromins--suspensions of the etiologic agent of Hansen's disease, Mycobacterium leprae--have been used as adjuncts to clinical parameters for classification in endemic areas. However, these have not been systematically studied in the United States. This report describes the results obtained from skin testing 38 volunteers (22 patients and 16 uninfected persons) with standard lepromin preparations. These results support the adjunctive value of lepromins for clinically classifying Hansen's disease in our "hypoendemic" population.

Hypnozoites of Plasmodium simiovale.

Hypnozoites of Plasmodium simiovale were detected in liver biopsies from a rhesus monkey (Macaca mulatta) inoculated eight days previously with sporozoites from heavily infected anopheline mosquitoes. The tissue forms, 6 mu in diameter, were found within the cytoplasm of hepatic parenchymal cells by immunofluorescence and restained with Giemsa. This is the first report of latent, pre-erythrocytic stages from an ovale-type relapsing malaria.

An ultrastructural study of the exoerythrocytic schizonts of Plasmodium cynomolgi and P. knowlesi in Macaca mulatta.

Exoerythrocytic schizonts of Plasmodium cynomolgi and P. knowlesi were examined by electron microscopy in biopsy samples of primate livers. With maturity the parasitophorous vacuole membrane becomes highly sculptured by the addition of a discontinuous dense thickening, the distribution of which can be a distinguishing character between these two species. The parasitophorous vacuole membrane follows the contours of the parasite faithfully with a minimal surrounding vacuole. The marked destruction of the cytoplasm of the host hepatocyte by most of the parasites studied however gave the distinct, but erroneous, appearance of a large parasitophorous vacuole at the light microscope level. The mature parasite often exhibited a highly invaginated surface contour with the result that the cytoplasm of the host cell and parasite became intimately interdigitated, this interweaving is unlikely to be recognized in light microscopic studies.

The hypnozoite and malarial relapse.

As described above, perhaps the most compelling arguments against the cyclic schizogony (Shortt-Garnham) hypothesis of malarial relapse have been (1) its inability to explain the disparate relapse patterns of P. vivax strains as they occur in nature, and (2) the dearth of concrete evidence for its support. The hypnozoite theory, in contrast, both provides an explanation for the variety of observed relapse patterns, and has direct morphologic and time-related, quantitative evidence for its support. Moreover, it provides a potential, unifying framework for the development of relapsing malaria from the time of introduction of sporozoites onward, taking as controlling factors, both the survival of the parasite and ecological interactions within its natural environment. It also suggests the possibility of an improved taxonomic classification, one based on whether or not the hypnozoite stage exists in a given species. Finally, Figure 6 presents a composite of relapse data pertinent to a number of strain groups of P. vivax, in comparison with a generalized, nonrelapsing species, schematically depicted within the framework of the hypnozoite theory. Thus, no hypnozoites are shown to result from sporozoites of the nonrelapsing species at one extreme, whereas no sporozoites undergoing immediate schizogony (i.e. without dormancy) are found at the other, the "lost" P. vivax strain of Nicolaiev. In between, varying proportions of sporozoites are depicted as producing hypnozoites, which exhibit varying periods of dormancy, ranging from less than 1 month (within the wide complement of the "tropical" strains) to approximately 21 months or more for the "northern" strains, before activation to schizogony and resultant relapse at the observed intervals. Although the actual proportions of each sporozoite/hypnozoite type within the strains depicted are unknown, a single, successful sporozoite can yield a parasitemic relapse--their distribution among the major strain groupings appears reasonably distinct, and, to some extent, defines each grouping. The sequences of sporozoite to pre-erythrocytic schizont, to merozoite release, to erythrocyte, as seen in nonrelapsing malarias and early primary parasitemias, versus sporozoite to hypnozoite, to pre-erythrocytic schizont, to erythrocyte, as seen in relapses, are easily inferred from this summary of observed relapses. It is difficult to see how these observed phenomena can be satisfactorily explained by a cyclic schizogony mechanism. It must be emphasized, nevertheless, that additional work with other relapsing and nonrelapsing species is necessary in order to establish the concept fully, and to determine the molecular mechanisms for dormancy and activation of hypnozoites.(ABSTRACT TRUNCATED AT 400 WORDS)

Observations on early and late post-sporozoite tissue stages in primate malaria. V. The effect of pyrimethamine and proguanil upon tissue hypnozoites and schizonts of Plasmodium cynomolgi bastianellii.

Two rhesus monkeys were each infected with 2.1 x 10(6) sporozoites of Plasmodium cynomolgi bastianellii; one was treated with 1.0 mg of pyrimethamine base per kg body weight for 5 d after sporozoite inoculation. A further 2 monkeys were each infected with 9.75 x 10(6) sporozoites of the same parasite; one was treated with 10 mg of proguanil per kg body weight for 4 out of 5 d after inoculation. The treated monkeys showed a delayed primary parasitaemia and relapses. In sections of liver biopsies taken 7.5 d after sporozoite inoculation, all monkeys showed numerous hypnozoites. However, there were no full grown schizonts and only rare retarded schizonts in the treated monkeys, in contrast to the untreated monkeys which had many mature or nearly mature schizonts. Later biopsies confirmed the continued presence of hypnozoites in all monkeys.

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.

Preliminary study of a Mycobacterium leprae bacterin vaccine in a human volunteer population in a nonendemic area.

Seventeen volunteers who had never resided in areas with significant transmission of leprosy (Hansen's disease) were inoculated intradermally with 1.5 X 10(8) killed, purified armadillo-derived Mycobacterium leprae in a standardized preparation being tested for possible vaccine use. The peak of local skin responses, consisting of induration with or without ulceration similar to the post-lepromin Mitsuda reaction, occurred between the 21st and 28th days after injection. The maximum mean induration diameter was 17.2 mm, the minimum, 6.1 mm. Sera tested with the ELISA technique revealed no humoral response to either the sonicated bacilli, M. leprae-specific phenolic glycolipid I antigen, or three semi-synthetic analogues reactive with lepromatous patients' sera. The dose of M. leprae inoculated appeared to be safe and without unacceptable reactions at the injection site. One volunteer developed a generalized skin rash parallel to the local reaction, but the relationship of the former to the inoculation was unclear.

Observations on early and late post-sporozoite tissue stages in primate malaria. IV. Pre-erythrocytic schizonts and/or hypnozoites of Chesson and North Korean strains of Plasmodium vivax in the chimpanzee.

In a continuing reexamination of plasmodial tissue stages within the context of the hypnozoite theory of malarial relapse, 2 strains of Plasmodium vivax with distinct and disparate relapse characteristics in humans were studied in chimpanzees. Following intravenous inoculation of massive numbers of salivary gland sporozoites, both the frequently relapsing Chesson strain and a North Korean strain characterized by predominantly delayed relapses exhibited relapse patterns and antimalarial sensitivity in the splenectomized chimpanzee essentially indistinguishable from those seen in humans. Examination of hepatic biopsies obtained at 7 and 10 days after infection revealed both pre-erythrocytic (pre-e) schizonts and hypnozoites in tissue obtained from the animal infected with the Chesson strain, but only rare hypnozoites (no pre-e schizonts) at 7 days in the animal infected with the North Korean strain. These findings, combined with the comparability of relapse behavior--which indicates the suitability of the chimpanzee as a model for the natural (human) host-parasite relationship--are essentially as predicted by the hypnozoite theory, despite the small numbers of tissue forms seen. Pre-erythrocytic schizogony of the Chesson strain in the liver was essentially indistinguishable from that of other strains studied, also underlining the suitability of this model system for tissue stage studies of P. vivax.

Studies on the North Korean strain of Plasmodium vivax in Aotus monkeys and different anophelines.

Twenty-two Aotus monkeys of different karyotypes were infected with the North Korean strain of Plasmodium vivax. Aotus lemurinus griseimembra animals from Colombia produced higher maximum parasitemias and more readily infected mosquitoes than did Aotus monkeys from Bolivia (K-VI) or Peru (K-V and K-X). Comparative feedings indicated that the most susceptible mosquito species was Anopheles stephensi, followed by An. gambiae, An. dirus, An. freeborni, An. quadrimaculatus, An. culicifacies, and An. maculatus.

Observations on early and late post-sporozoite tissue stages in primate malaria. III. Further attempts to find early forms and to correlate hypnozoites with growing exo-erythrocytic schizonts and parasitaemic relapses in Plasmodium cynomolgi bastianellii infections.

Rhesus monkeys were heavily infected with sporozoites of Plasmodium cynomolgi bastianellii in an attempt to demonstrate the site of invasion of sporozoites into tissue cells and their growth there. Further attempts were made to correlate the appearance and loss of hypnozoites with parasitaemic relapses. Hypnozoites were demonstrated and once again shown to decrease in numbers over 229 days during which time the infection showed parasitaemic relapses. Liver biopsies taken at two-day intervals for 12 days showed that hypnozoites decreased in numbers over-all and growing schizonts were demonstrated in the liver. At this time a parasite the size of a hypnozoite was seen with two nuclei and another was seen with an elongate, possibly dividing nucleus in one monkey. an attempt to find the location of the early intracellular exoerythrocytic forms in the liver at various times less than 40 hours after infection using smears and immunological staining with newly prepared anti-sera failed. Large numbers of sporozoites of P. knowlesi were also injected into a rhesus monkey the liver of which on the fifth day after infection showed no hypnozoites among 157 sections of growing schizonts and no parasites at all on the 42nd day after infection. In P. cynomolgi bastianellii infections parasites, mostly hypnozoites, were found in the liver up to 229 days after infection.

Loiasis and renal failure.

A patient with a filarial infection due to Loa loa and renal failure was treated with a modified regimen of diethylcarbamazine while receiving hemodialysis. Infection with Loa loa may be associated with glomerulonephritis due to immunologically mediated injury. Serum immune complexes were elevated in our patient, and a kidney biopsy specimen revealed globally sclerosed glomeruli. As gauged by the clearance of the microfilaremia, the treatment regimen proved safe and effective.

Enzootic schistosomiasis in a Louisiana armadillo.

Trematode eggs compatible in characteristics with those of Heterobilharzia americana, a probable cause of human schistosomal dermatitis in the Gulf States of the U.S., were found in the cirrhotic liver and other viscera of a wild-caught armadillo in Louisiana. Although adult worms were not found, the current observation suggests a need to extend the known host range of this common schistosome parasite of carnivores and other wild animals in the South, and may have additional implications in the context of the public health importance of the armadillo.

Identification of hypnozoites and tissue schizonts of Plasmodium vivax and P. cynomolgi by the immunoperoxidase method.

An immunoperoxidase technique was used to detect hypnozoites and liver schizonts of the primate malaria species Plasmodium vivax and P. cynomolgi bastianellii in Carnoy's-fixed sections. Anti-P. cynomolgi serum and a peroxidase-conjugated anti-monkey IgG serum rendered 7-day pre-erythrocytic forms clearly visible. The technique retains the specificity of the immunofluorescence method while having the advantage of a permanent preparation. Detection of hyponozoites by this alternative method provides further evidence for their plasmodial nature.

Timing of stages and changes in numbers of multiply-infected erythrocytes during the asexual cycle of Plasmodium cynomolgi bastianellii.

Previous evaluations of the timing of developing stages in the erythrocytic cycle of the simian malaria parasite, Plasmodium cynomolgi, a frequently used model for relapsing malaria in man, have depended on procurement of blood samples at 4- or 6-hourly intervals. This brief report describes the blood cycle as determined from hourly bleedings, thus providing a more discriminating sequence usable for immunochemical or biochemical study of specific stages. The approximate timing of development for five defined stages in the asexual cycle was determined to be 12-14 hours for young trophozoites, 22-24 hours for developing trophozoites, 5-7 hours for mature trophozoites, 5-6 hours for early schizonts, and 3-5 hours for late schizonts. It was also demonstrated that the rate of erythrocytic multiple infection declined in proportion to the maturity of the parasite. Young and developing trophozoites presented the most numerous cases of multiple infection whereas very few schizonts were seen multiply infecting a host cell.

Electrophoretic and immunoelectrophoretic study of the enzymatic degradation of human orosomucoid (alpha 1 -acid glycoprotein).

Purified, intact orosomucoid (alpha 1-acid glycoprotein) derived from whole human plasma was incubated with a number of proteolytic and saccharolytic enzymes under a variety of conditions. The unfractionated digests were immediately examined by both agar gel- and immunoelectrophoresis for the presence of antigenically active (precipitating) and/or inactive macrofragments. Despite otherwise clear evidence of rapid degradation by several proteases, no antigenic subunits were detected. Among the glycosidases, almond emulsin produced a suggestion of modification of the carbohydrate moiety of a sialic acid-poor orosomucoid preparation obtained from Cohn Fr. VI, but had no effect on antigenic properties. These results are presented as further evidence for a lack of involvement of the extensive carbohydrate component of orosomucoid in its antigenic reactions, and support previous data implicating the polypeptide chain as solely responsible for its antigenicity.