Lumen phase specific cross immunity between Hymenolepis microstoma and H. nana in mice

, and 1988. Lumen phase specific cross immunity between Hymenolepis microstoma and H. nana in mice. International Journal for Parasitology 18: 1019–1027. When mice inoculated with five cysticercoids of Hymenolepis microstoma were challenged with H. nana, they showed strong resistance to challenges with both eggs and cysticercoids of H. nana from day 20. The immunity became complete from day 30 onward: no tissue cysticercoids or lumenal adults of H. nana were established in these mice. However, when mice were challenged with H. nana 10 or 20 days after 10-day old immature H. microstoma were removed by an anthelmintic, the immunity evoked was directed exclusively against the lumenal phase of the cysticercoid challenge but not the tissue cysticercoids of the egg challenge. When mice experienced the prepatent infection with H. microstoma twice, the immunity evoked was also against the lumenal phase of the egg challenge: the oncospheres developed into tissue cysticercoids but thereafter completely failed to develop into lumenal adult tapeworms. Infection with a single cysticercoid of H. microstoma was shown to be sufficient to evoke immunity against H. nana cysticercoid infections in two strains of mice. Sera from mice which experienced a patent infection with H. microstoma revealed that IgG, IgA, IgM isotypes reacted against oncospheres and cysticercoids of both species, while sera from mice which experienced two prepatent infections reacted with cysticercoids only. Sera from H. microstoma infected mice resistant to H. nana caused precipitations on 4-day-old H. nana in vitro. A correlation exists between the presence of stage specific antibodies and resistance to the different stages.     

Primary infection with mouse-derived cysticercoids of Hymenolepis nana prepared from baby or adult mice and secondary infections with eggs or cysticercoids

Oral infection with mouse-derived cysticercoids (cysts) of Hymenolepis nana on day 0 did not make the 5 ± 1 week old mouse host immune to egg challenge by day 7 of the prepatent period, although the number of cyst-derived tapeworms was 1000 times greater than that of egg-derived tapeworms sufficient to make the host immune by day 7. Neither cysts recovered from immunologically competent 5 ± 1 week old donor mice, which should have become immune within 2 days of egg inoculation, nor those from incompetent 5–7 day-old baby mice given eggs when 0–2 days old made the host immune. The time course of differentiation of cysts in baby mice was not different from that in 5 ± 1 week-old mice. Mice infected twice with cysts on days 0 and 4 did not become immune either. Rapid protective immunity against egg challenge was acquired by inoculation exclusively with eggs but not with cysts. Apparently cysts differ from oncospheres in their immunogenicity. The importance of cysts for analysing the mouse—H. nana system from the immunological point of view is discussed.     

Primary alveolar echinococcosis: Course of larval development and antibody responses in intermediate host rodents with different genetic backgrounds after oral infection with eggs of Echinococcus multilocularis

We investigated parasite establishment, subsequent larval development and antibody responses in gerbils, cotton rats and 4 inbred mouse strains until 16 weeks post inoculation (p.i.) with 200 eggs of Echinococcus multilocularis. The rate of parasite establishment in the liver determined at 4 weeks p.i. was highest in DBA/2, followed by AKR/N, C57BL/10 and C57BL/6 mice, whereas gerbils harboured few parasite foci. The accurate number of liver lesions in cotton rats could not be determined due to rapid growth and advanced multivesiculation of the parasite observed at 2 weeks p.i. The course of larval development was most advanced in DBA/2 mice with mature protoscolex formation at 16 weeks p.i., followed by AKR/N harbouring metacestodes with sparsely distributed immature protoscoleces. On the other hand, C57BL/6 and C57BL/10 mice had infertile metacestodes without any protoscolex formation. The parasite growth in mice was totally slower than those in gerbils and cotton rats. Specific IgG and IgM responses against 3 types of native crude antigens of larval E. multilocularis were evaluated using somatic extracts of and vesicle fluid of metacestode, and somatic extracts from purified protoscoleces. The 4 mouse strains demonstrated basically similar kinetics with apparent IgG and IgM increases at 9 weeks p.i. and thereafter, except C57BL/10, exhibited higher levels of IgM against crude antigens at some time point of infection. On the other hand, a follow-up determination of specific IgG and IgM levels against recombinant antigens from larval E. multilocularis revealed that each mouse strain showed different antibody-level kinetics. The findings in the present study demonstrate that the course of host–parasite interactions in primary alveolar echinococcosis, caused by larval E. multilocularis, clearly varies among intermediate host rodents with different genetic backgrounds.     

A Hospital-Based Serological Survey of Cryptosporidiosis in the Republic of Korea

The seroprevalence of cryptosporidiosis was examined using patients'' sera collected from hospitals located in 4 different areas of the Republic of Korea. ELISA was used to measure antibody titers against Cryptosporidium parvum antigens from a total of 2,394 serum samples, which were collected randomly from patients in local hospitals; 1) Chungbuk National University Hospital, 2) Konkuk University Hospital, 3) local hospitals in Chuncheon, Gangwon-do (province), 4) Jeonnam National University Hospital, from 2002 through 2003. Of the 2,394 samples assayed, 34%, 26%, and 56% were positive for C. parvum-specific IgG, IgM, and IgA antibodies, respectively. Positive IgG titers were most common in sera from Jeonnam National University Hospital, Gwangju, Jeollanam-do, and positive IgM titers were most common in sera from Chungbuk National University Hospital, Cheongju, Chuncheongbuk-do. The seropositivity was positively correlated with age for both the IgG and IgA antibodies but was negatively correlated with age for the IgM antibodies. Western blotting revealed that 92%, 83%, and 77% of sera positive for IgG, IgM, and IgA ELISA reacted with 27-kDa antigens, respectively. These results suggested that infection with Cryptosporidium in hospital patients occurs more commonly than previously reported in the Republic of Korea.     

Echinococcus multilocularis: distribution and persistence of specific host immunoglobulins on cysts membranes

Cyst vesicles were obtained from larval cyst masses of Echinococcus multilocularis grown in Medium 858 in vitro or isolated from the intraperitoneal or subcutaneous larval cyst mass of C57BL/6J mice infected 12 weeks previously. Antigenic determinants were present on the outermost section of the laminated layer and throughout the germinal layer of the cysts. Specific host antibodies of the IgG1, IgG2a, IgG2b, and IgM classes and complement component C3 but not host IgA or C-reactive protein were detected on the intact cysts by the indirect immunofluorescent technique. Specific antibodies were bound to the epitopes on the laminated layer but were not detected on the germinal layer. Host albumin, however, was found on the laminated layer, germinal layer, and within the intact cyst. IgG2a and IgG2b were high-affinity antibodies but IgG1 and IgM were low-affinity antibodies as they were eluted easily from the laminated layer with two washes of sodium phosphate buffer (0.01 M, pH 7.2) containing 0.15 M NaCl. The significance of bound antibody in complement activation and antibody-dependent cell-mediated cytotoxicity of the proliferative phase (cyst) of alveolar hydatid cyst is discussed.     

Intestinal Resistance in the Experimental Enteric Infection of Mice with a Mouse Adenovirus

In order to find out whether the mouse adenovirus-neutralizing substance, which appeared in the intestinal tract of mice orally infected with mouse adenovirus, was an immunoglobulin, examinations were carried out for the status of 3 classes of immunoglobulin, IgA, IgG, and IgM, in the intestinal tract as well as in the serum of the mouse. In infected mice, as in uninfected mice, the serum contained much IgG, a moderate amount of IgA, and a small amount of IgM, whereas the intestinal wall showed a moderate amount of IgA, a small amount of IgG and no IgM, and the intestinal contents contained a moderate amount of IgA. Secondly, DEAE-cellulose chromatography or Sephadex G-200 gel filtration was done in order to know whether the virus-neutralizing activity was recoverable in the fractions containing some class of immunoglobulin. The result indicated that a large part of the activity in the serum was recovered in the fractions of IgG and a small part in those of IgA. In the case of the intestinal wall, a large part of the activity was found in the fractions of IgA, and only a small part in the fractions containing both IgG and IgA. In the intestinal contents, the activity was detected solely in the fractions containing IgA. Finally, when the substance from the intestinal wall was purified by DEAE and Sephadex, a parallel increase of both IgA and the virus-neutralizing activity per protein content was observed. Thus, it became clear that the mouse adenovirus-neutralizing substance in the intestinal tract was an antibody against the virus, and that it mostly belongs to IgA.     

Enzyme-linked immunosorbent assay (ELISA) for IgM, IgG and IgA class antibodies against Candida albicans antigens: development and comparison with other methods

An extract of Candida albicans was used as an antigen on microtitre plates in the enzyme-linked immunosorbent assay (ELISA) to measure IgM, IgG and IgA class antibodies in the sera of hospitalized patients. It was found that of these patient sera that reacted positively in Ouchterlony immunodiffusion (ID) when undiluted, 58% were also positive in the ELISA against the same antigen preparation. However, all the sera with an ID titre of 1:2 or higher were ELISA-positive, demonstrating especially IgG and IgA. Of the sera positive by counterimmunoelectrophoresis against somatic and metabolic antigens of C. albicans, 86% were positive by ELISA. Reactions in precipitin-negative sera, if they occurred, usually demonstrated IgM or IgA. The sera with high passive haemagglutination or indirect immunofluorescence titres against surface antigens of C. albicans were positive in the IgG and IgA assays, while approximately one third were positive in the IgM assay.     

Murine T cell clones specific for Hymenolepis nana: generation and functional analysis in vivo and in vitro.

To examine the role of the T cell in protective immunity to Hymenolepis nana, H. nana-specific clonal lymphocytes were generated from mesenteric lymph nodes of BALB/c mice infected with H. nana, and some of their functions were analyzed in vitro and in vivo. Following limiting dilution techniques, five clones were generated from mesenteric lymph node cell populations. All of these clones expressed the L3T4⁺, Lyt-2.2⁻ phenotype and proliferated in vitro in response to soluble egg antigen of H. nana. Of five clones, three secreted interleukin 2 (IL-2) and interferon-γ (IFN-γ) after stimulation with egg antigen. Furthermore, these three clones conferred local delayed-type hypersensitivity to egg antigen. The remaining two clones produced interleukin 4 (IL-4) in response to egg antigen, and could not mediate local delayed-type hypersensitivity. Adoptive transfer experiments using clonal lymphocytes were also undertaken in an attempt to define cell types involved in protective immunity. Clonal lymphocytes secreting both IL-2 and IFN-γ transferred protective immunity, equivalent to that obtained by non-cultured-sensitized mesenteric lymph node cells. They were effective in very small numbers. However, clonal lymphocytes that secreted IL-4 did not transfer protective immunity. These results suggest that helper T lymphocytes, especially the Th1 subtype, are involved in protective immunity against H. nana.     

Trypanosoma musculi co-express several receptors binding rodent IgM, IgE, and IgG subclasses

The present work demonstrates the expression of receptors for the Fc portion of rodent Ig by the murine parasite Trypanosoma musculi. By using a rosette assay adapted to the parasite morphology and by flow cytometry analysis, three distinct receptors were identified. A receptor binding rabbit or rat polyclonal IgG and mouse monoclonal IgG1, IgG2a, and IgG2b was found on parasites purified from the blood and the peritoneal cavity of infected mice and on parasites maintained in culture conditions. This IgG receptor was degraded by pepsin. A separate receptor, binding only mouse monoclonal IgG3 was observed on cultured parasites. A receptor binding rabbit, rat, and mouse IgM was found on cultured and peritoneal parasites, but not on blood parasites. This receptor did not bind IgG or IgA but it bound mouse and rat IgE as well as IgM. It was degraded by trypsin. IgG and IgM/IgE receptors were co-expressed on single parasites. They were not of host origin but synthesized by trypanosomes as shown by reexpression in vitro after proteolytic degradation. Their expression was variable with the development of trypanosomes both in vitro and in vivo.     

Study of the antigenic relationship between animal and human immunoglobulins using antisera to human immunoglobulins]

Cross-reactivity of monospecific antisera to human immunoglobulins with animal sera of 10 species was studied by immunoelectrophoresis and radial immunodiffusion. Antisera to IgG were shown to reveal IgG of all the species studied, antisera to IgM and especially to IgA cross reacted less extensively. The greatest number of cross reactions were given by the antisera obtained as a result of hyperimmunization. Hyperimmune monospecific antisera to human IgG, IgA, and IgM can be used for the identification of animal immunoglobulins during their isolation from the sera and for their quantitation by radial immunodiffusion.     

Echinococcus multilocularis: Immunoglobulin and antibody response in C57BL6J mice

Each of 50 male C57BL/6J mice was infected intraperitoneally with 50 cysts of Echinococcus multilocularis. At 2, 4, 6, 8, and 14 weeks after infection, 10 mice were sacrificed, their larval cyst masses weighed, and their sera collected. Each serum sample from uninfected control and infected mice was adsorbed twice with two batches of E. multilocularis antigen conjugated to Sepharose beads. The concentrations of IgG1, IgG2a, IgG2b, IgM, and IgA in unadsorbed and IgG1, IgG2b, and IgM in adsorbed sera were quantified by the radial immunodiffusion technique. Hydatid mice produced increasingly large amounts of IgG1 and IgM; small measurable increases of IgG2b and no significant increases of IgG2a and IgA were observed during the course of infection. During the rapid growth phase of the cysts (6 to 14 weeks) IgG1 antibodies were found to range from 86 to 93% and IgM antibodies from 17 to 33% of the total IgG1 and IgM. However, the actual protein concentrations of IgM antibodies (761 and 1215 mg/dl) were higher than the sum of the protein concentrations of IgG1 and IgG2b antibodies (411 and 779 mg/dl). The significance of the relative concentrations of IgM, IgG1, IgG2a, and IgG2b antibodies is discussed with reference to their effectiveness in antibody-dependent cellular cytotoxicity and complement-mediated lysis in the control of alveolar hydatid disease.     

Mannose and the ‘Crowding effect’ of Hymenolepis in rats

Hymenolepis diminuta from rats infected with 10 cysticercoids and fed on a diet containing 3% (w/w) mannose for 4 weeks were found to be, on average, much heavier in terms of dry weight (46 mg) than those from rats fed on diets containing an equivalent concentration of either galactose, glucose or fructose (18 mg). Subsequently, the numbers, egg production and dry weights of worms were determined from rats which had been infected with doses varying from 0 to 160 cysticercoids per rat and fed on diets containing either 0, 1, 4, or 8% mannose (w/w). Density-dependent decreases in both worm dry weight and egg production were detected at 5 weeks post-infection, but both the number of worms recovered and their prepatent period appeared to be independent of cysticercoid dose. Differences in the distribution pattern of individual worm dry weights were observed between rats harbouring low (1–15) and high (16–135) worm burdens. Worms recovered from rats infected with 10 cysticercoids and fed on a diet containing 4% mannose (w/w) for 3 weeks were found to be, on average, more than twice as heavy (37 mg) as those from rats fed on diets containing 1, 2, or 8% mannose (w/w) for equivalent periods of time (16 mg). The results indicate that the ‘crowding effect’ cannot be explained simply in terms of inter-worm competition for carbohydrate.     

Development of monoclonal IgA and an apparent IgG in a patient with macroglobulinemia: sharing of individually specific antigenic determinants among IgM, IgA, and IgG.

Patient CM, who initially was diagnosed as having macroglobulinemia (IgM, kappa) was subsequently found to develop a monoclonal IgA(kappa) protein. Rabbit antisera directed against the patient's IgAm and IgM were rendered specific for individual antigenic (ind) determinants. The anti-IgAm and IgM ind sera reacted with both 131I labeled monoclonal proteins in a double antibody radioimmunoassay (RIA). In addition, both monoclonal immunoglobulins inhibited the reaction between labeled immunoglobulin and both ind antisera, and statistical analysis of the data suggested that the shared ind determinants were identical. The IgG fraction of patient CM's serum also contained a component which competed with both monoclonal IgA (CM) and IgM (CM) in the RIA specific for ind determinants. Analysis of serum samples taken over a 2-year period revealed that, in addition to IgM, both the IgA and IgG components possessing the shared ind determinant(s) were present in low concentrations in the earliest sample, although not detected by conventional techniques. The monoclonal IgA and the IgG component were found to increase in concentration over this time interval with a concomitant decrease in IgM. The regulation of immunoglobulin expression with respect to the proposed models of gene organization in antibody-producing cells was discussed.     

Immunological follow-up of hydatid cyst cases

Hydatid disease is caused by Echinococcus granulosus. In this study, we aimed to investigate the benefit of monitoring cases with hydatid cyst by means of immune components in patients in a long-term follow-up after surgery. Eighty-four preoperative and postoperative serum samples from 14 cases undergoing surgery for hydatid disease were evaluated in terms of immune parameters, such as total and specific IgE, IgG, IgM, IgA and complement. Total and specific IgE were determined by ELISA. Specific IgG levels were measured by indirect hemagglutination.Total IgG, IgM, IgA and complement (C3 and C4) were detected by nephelometry. Imaging studies were also carried out during the follow-up. In none of the patients hydatid cysts were detected during the follow-up. Total IgE levels in the sera of the patients decreased to normal six months after surgery. Although specific IgE against echinococcal antigens decreased one year after operation, levels were still significantly high. There were no changes in the levels of anti-Echinococcus IgG and total IgG in follow-up period. Additionally, other parameters, such as IgA, IgM, C3 and C4, were not affected.     

Carbon,nitrogen and phosphorus stoichiometry of Myrica nana in Yunnan,China

Aims Carbon (C), nitrogen (N) and phosphorus (P) play important roles in plant growth and physiological functions. We aimed at exploring the intrinsic relationships of C, N and P in Myrica nana—a common shrub in Yunnan Province—as well as their relationships with pant biomass and soil nutrients.
Methods We measured the concentration of C, N and P of M. nana from 29 sites for their magnitudes and correlations with soil nutrients.
Important findings 1) The arithmetic mean value of C, N and P concentration in the roots, stems and leaves of M. nana was 45.94%, 0.54%, 0.03%, and 46.32%, 0.58%, 0.03%, and 49.05%, 1.70%, 0.06%, respectively. C, N and P concentrations in the leaves were significantly higher than those in the roots and the stems. The C:N:P in roots, stems and leaves was 1531:18:1, 1544:19:1, and 818:10:1, respectively. 2) The C concentration and N:P in leaves of M. nana decreased with the increase of biomass of M. nana; the leaf C concentration was significantly correlated with biomass (p < 0.01), while the correlation between N:P and biomass was not significant (p > 0.05). The leaf N increased with the increase of plant biomass, the P was significantly correlated with biomass (p < 0.05), but the correlation between N concentration and biomass was not significant (p > 0.05). N:P in leaves was 34.2, suggesting that plant growth was limited by P. 3) C, N and P concentration in the roots were significantly correlated with soil P (p < 0.05), with N, P concentrations correlated with soil P positively (p < 0.01) and C negatively (p < 0.05). C concentration in the stems was significantly and negatively correlated with soil C, N, with significant correlation with C, N, and P concentration (p < 0.01). P concentration in the stems was significantly and positively correlated with soil P concentration (p < 0.01), while leaf P significantly and positively correlated with soil C, N and P (p < 0.01); leaf C concentration was significantly and negatively correlated with soil P (p < 0.01).     

Hymenolepis nana: Maturation in an immunosuppressed unnatural rat host

When eggs of the dwarf tapeworm Hymenolepis nana, cycled exclusively and directly through mice for more than 10 years, were inoculated into previously uninfected inbred Fischer (F344) strain rats, they failed to mature in the rat intestinal lumen. Eggs of H. nana inoculated into the rat developed normally into cysticercoids (cysts) in the intestinal tissue, but thereafter failed to mature in the lumen except when the host was treated with cortisone acetate from the day of cyst maturation. The Fischer rat initially given eggs of H. nana became completely immune to egg challenge within 2 days of egg inoculation; no cysts derived from challenge eggs were found in the immunized rat. Immunosuppression, assessed by the success of cyst recovery in the tissue 4 days after egg challenge, had no promotive effect on the recovery of adult worms derived from eggs initially inoculated. Rats initially given eggs and immunosuppressed by cyclophosphamide or antithymocyte serum did not harbor any adult worms. Cortisone acetate treatment which was sufficient for eggs inoculated to mature (a total of 75 or even 200 mg, from Day 5 of egg inoculation) had no effects of immunosuppression, whereas cortisone acetate treatment which was sufficient for immunosuppression (a total of 150 mg from Day -2, two days prior to the initial egg inoculation) induced some adult formation as well. In addition, when mouse-derived cysts were inoculated into the rat instead of eggs, they also failed completely to mature even when the rat was treated with cyclophosphamide or antithimocyte serum. However, when the rat was treated with cortisone acetate from the day of cyst inoculation, the cysts developed into adult worms. Therefore, these results indicate that the Fischer rat clearly differs in its susceptibility to the tissue phase of egg inoculation and to the lumen phase of cyst inoculation of H. nana, and strongly suggest that the failure of maturation of H. nana in the unnatural host Fischer rat is not attributed to innate and/ or acquired immunity of the rat but to other nonimmunological mechanisms.     

The mode of passive protection against Hymenolepis nana induced by serum transfer

A protective immunity against the cestode Hymenolepis nana was transferred with serum taken from actively immunized mice. All of 17 pooled sera examined, which were taken from mice immunized for 3 or more weeks, were strongly effective. Intraperitoneal injection of a total of 3·0 ml serum made the recipient mice (4–5 weeks old) almost completely immune. In almost all the mice given immune serum no cysticercoids were found on day 4. In mice receiving immune serum, oncospheres hatched, invaded the intestinal villi and differentiated to stage II or III larvae, but failed to develop to fully developed cysticercoids. The degree of protection conferred by serum transfer was similar to, but slightly weaker than that stimulated by active immunization. The major effect of immune serum was damaging hatched oncospheres in both the intestinal lumen and the villi within 1 day post infection.     

Generation of monoclonal antibodies to Macaca mulatta (rhesus) IgA

One IgG1 and five IgM murine monoclonal antibodies (mAb) specific for rhesus (Rh) IgA were generated. These mAbs bound to Rh IgA but not IgG or IgM when tested by enzyme-linked immunosorbent assay. Immunoblotting revealed that the mAbs reacted with the alpha heavy chain of Rh but not human IgA. The IgG1 anti-Rh IgA mAb detected IgA-producing cells in sections of monkey gut examined by immunofluorescent staining. These mAbs should be useful for characterizing IgA responses in the Rh monkey.     

Local antibody response in Peyer's patches to the orally administered dietary protein antigen

To understand local antibody production to dietary protein antigens in the gut, the reactivity of the monoclonal antibodies (mAbs) from Peyer's patches of BALB/c mice raised against orally administered hen egg lysozyme (HEL) was studied. These mAbs were of IgG1 (7 clones), IgA (5 clones) and IgM (13 clones) isotypes. Some of the HEL-binding mAbs preferentially reacted with reduced, carboxy-methylated HEL, rather than with native HEL. MAbs of the IgA and IgM isotypes had cross-reactivity with other unrelated environmental antigens such as E. coli, single-strand DNA, and soluble components of mouse food. In contrast, the IgG1 mAbs did not cross-react with these antigens. The average of the Kd values for HEL of these mAbs was in the order of 10(-6) M, which is moderately higher than those of mAbs from the preimmune repertoire. These results suggest that, under normal physiological conditions, orally administered dietary proteins predominantly induce the local production of polyreactive IgA/IgM antibodies cross-reacting with environmental luminal antigens.     

Humoral antibody response to cattle to formalinized Staphylococcus aureus vaccine.

Five cows were inoculated intradermally with formalinized Staphylococcus aureus suspension in Freund's complete adjunvant and the development of the humoral antibody response was followed as judged by the agglutination titer of sera, at various intervals post inoculation. Highest titers were observed at 78-87 days post inoculation. Agglutinating activity was found in IgM and IgG fractions (IgG1 and IgG2) of both serum and colostrum. The agglutinating activity of colostrum was significantly higher at 12 than at 24 and 36 h, post partum. However, no such activity was detected in either normal cow serum or colostrum against S. aureus.