Strongyloides stercoralis host-adapted third-stage larvae are the target of eosinophil-associated immune-mediated killing in mice

Host-adapted, transformed, Strongyloides stercoralis third-stage larvae (L3+) were previously found to be antigenically different from free-living, infective, third-stage larvae (L3). These antigenic differences were reproduced by transformation of free-living larvae in tissue culture medium at 37 C over 24 hr. Transformed L3 of both derivations were given as challenge infections in diffusion chambers to naive mice and mice immunized with S. stercoralis L3. Within 12 hr, the challenge infections were killed regardless of whether the L3+ were generated in vitro or in vivo. Eosinophils, previously found to be important in the immune response to S. stercoralis larvae, were recruited into the L3+ microenvironment within 12 hr of challenge infection in immune mice, which supports the previously proposed mechanisms of S. stercoralis larval killing. Thus, S. stercoralis L3+ appear to be targets of the immune response in mice instead of being involved in immune evasion.     

Portrait of inequality

BALB/c mice immunized with L3 of Brugia pahangi, irradiated at 45 kRad from a 137Caesium source, are strongly immune to challenge infection (75-100% reduction in the recovery of challenge infection larvae on day 6 post-challenge). The target of immunity appears to be the post-infective L3, as challenge infection larvae are killed within 5-6 days of infection. By immunoblot analysis, serum from immune animals recognizes a limited set of somatic antigens, the majority of which are shared between different life cycle stages. Serum from immune mice also strongly recognizes larval surface antigens by immunofluorescence, some of which may be stage specific. The larval surface determinants do not appear to be protein or glycoprotein by standard immunochemical analysis. A proportion of the antibody response of the BALB/c mouse is directed towards phosphorylcholine epitopes on filarial antigens, but the limited antigen recognition cannot be explained on the basis of the mouse strain used, as CBA/Ca mice recognize a similar limited set of antigens.     

Brugia pahangi: effects of protective resistance on lymphatic lesions and granulomatous inflammation in infected jirds (Meriones unguiculatus)

Effects of protective resistance on lymphatic lesions and granulomatous inflammation in infected jirds (Meriones unguiculatus). Experimental Parasitology 77, 395-404. The hypothesis that protective immune responses play a role in the induction of filarial-associated lymphatic lesions was tested in jirds immunized twice with 75 Brugia pahangi radiation-attenuated third-stage larvae. Lymphatic lesions and granulomatous reactivity were compared in immunized, infected, and naive jirds at both acute and chronic periods following challenge with 100 third-stage larvae. Challenge worm burdens were reduced in immunized jirds at both infection periods. The ratio of lymph thrombi to lymphatic worms, an indicator of lymphatic lesion severity, was significantly greater in immunized jirds than in nonimmunized-challenged jirds during acute but not chronic infections. Parasite-specific-granulomatous hypersensitivity was assessed by measurements of granuloma areas around B. pahangi-soluble adult worm antigen-coated sepharose beads embolized in the lungs prior to necropsy. Marked granulomatous inflammatory responses seen during the acute period in both immunized-challenged and nonimmunized-challenged jirds were significantly reduced in similar jirds during chronic periods. Jirds with existing B. pahangi infections were not resistant to homologous challenge infection and had fewer lymphatic lesions and reduced granulomatous inflammatory responses to soluble adult worm antigen compared to previously naive jirds at acute periods postchallenge. These data suggest that protective immune responses increase the severity of filariae-induced lymphatic inflammation. The subsequent modulation of these lesions is probably associated with parasites that survived the protective immune response.     

The clinical picture and dynamics of long-term late remissions in the outcome of shift-like schizophrenia]

Varying doses of protoscolices of Echinococcus granulosus were injected intraperitoneally into Mongolian jirds, Swiss mice, and golden hamsters. After 4 months the animals were killed and the numbers of developing cysts counted. Jirds were by far the most susceptible hosts. Doses of 500 protoscolices produced 100% infection rates in both weanlings and adults. Comparable infection rates in weanling mice required inocula of 5,000 protoscolices, but even at this dose hamsters were refractory to infection. Three of 5 jirds developed pulmonary cysts after intravenous administration of 500 protoscolices. Serologic responses in infected jirds were followed using the indirect hemagglutination tests and a purified fraction of hydatid cyst fluid (HCF) as antigen. Titers reached a plateau after 15 weeks and were maintained for several months. Much brisker serologic responses occurred in animals sensitized with the antigenic fraction in various adjuvant vehicles. At no time during infection did jirds show any evidence of immediate hypersensitivity to HCF in direct skin tests, passive cutaneous anaphylactic tests, or after intravenous challenge with antigen. After artificial immunization with hapten-conjugated antigens or HCF, jirds underwent a fatal shock following intravenous challenge with antigen. HCF provoked the appearance of circulating antibodies which were capable of sensitizing normal recipients for passive systemic anaphylaxis after a latent period of 72 hr. It is concluded that the failure of the infected jird to develop immediate hypersensitivity responses to E. granulosus represents a marked deviation from the parrern of the immune response in echinococcosis in man and domestic animals and must be considered in the future use of this animal in experimental studies on the host-parasite relationship.     

Strongyloides stercoralis: role of antibody and complement in immunity to the third stage of larvae in BALB/cByJ mice

Mice immunized against Strongyloides stercoralis L3 were shown to kill greater than 90% of challenge larvae contained within diffusion chambers. The objective of the present study was to identify the host components responsible for immunity. Serum from unprotected, control mice and protected, immune mice in doses of 25-500 microliters was transferred into naive mice at the same time and location as larval challenge. Transfer of as little as 50 microliters of immune serum was able to confer protective immunity. The serum-transferred immunity was ablated by excluding cells from the larval microenvironment or by depleting granulocytes through monoclonal antibody treatment in the recipient mice. Specific antibody isotypes were isolated using protein G and isotype-specific affinity columns. The resulting transfer experiments identified IgM as the isotype responsible for protective immunity to S. stercoralis L3. Antibody binding studies in vivo were performed and only IgM bound to the surface of infective L3 and host-derived L3 (L3+) in immune animals. Elevated levels of C3 were also found bound to the surface of L3/L3+ in immune mice. Cobra venom factor treatment of immunized mice to deplete complement completely eliminated C3 binding to the surface of L3/L3+ and ablated immunity. Therefore, IgM, complement, and granulocytes are necessary for immune elimination of S. stercoralis L3/L3+. Identification of antigens recognized by IgM may help select possible vaccine candidates.     

Analysis of the Drosophila host defense in domino mutant larvae, which are devoid of hemocytes

We have analyzed the Drosophila immune response in domino mutant larvae, which are devoid of blood cells. The domino mutants have a good larval viability, but they die as prepupae. We show that, on immune challenge, induction of the genes encoding antimicrobial peptides in the fat body is not affected significantly in the mutant larvae, indicating that hemocytes are not essential in this process. The hemocoele of domino larvae contains numerous live microorganisms, the presence of which induces a weak antimicrobial response in the fat body. A full response is observed only after septic injury. We propose that the fat body cells are activated both by the presence of microorganisms and by injury and that injury potentiates the effect of microorganisms. Survival experiments after an immune challenge showed that domino mutants devoid of blood cells maintain a wild-type resistance to septic injury. This resistance was also observed in mutant larvae in which the synthesis of antibacterial peptides is impaired (immune deficiency larvae) and in mutants that are deficient for humoral melanization (Black cells larvae). However, if domino was combined with either the immune deficiency or the Black cell mutation, the resistance to septic injury was reduced severely. These results establish the relevance of the three immune reactions: phagocytosis, synthesis of antibacterial peptides, and melanization. By working in synergy, they provide Drosophila a highly effective defense against injury and/or infection.     

Reduced hexose transport by enterocytes associated with rapid, noninjurious rejection of Trichinella spiralis from immune rats

Rats immunized against Trichinella spiralis rejected larvae from a challenge infection within minutes. Infectivity of these once-rejected larvae for nonimmune rats following intestine-to-intestine transfer, infectivity for nonimmune mice following oral inoculation, and normal development in these mice indicated that the parasite did not sustain immediate or long term damage as a result of the host's immune response. Associated with rapid worm rejection was a change in host intestinal function - altered absorption. Uptake of a hexose, 14C-beta-methyl-D-glucoside, was studied in intestinal segments isolated from rats before and 30 min after primary and secondary infections. Larvae were administered intraintestinally. Absorption was measured using a tissue accumulation method, under conditions of substrate saturation (30 mMolar) and over a 5-min period. 3H-mannitol was used as a marker to measure extracellular water, hence, extracellular sugar. Following challenge infection of previously infected (immunized) hosts, there was a significant reduction (22%; P less than 0.05) in hexose absorption as compared to the uptake rate before challenge. In contrast, cellular uptake before and 30 min after primary infection was similar. These findings are presented in support of the hypothesis that the failure of T. spiralis larvae to infect immune hosts is the result, not of direct worm damage, but of functional changes in gut epithelium, the habitat of the parasite.     

Intestinal epithelial membrane changes in rats immune to Trichinella spiralis

Establishment of Trichinella spiralis infective larvae is blocked to a large degree in the immune rat as compared with the nonimmune host. The rapidity with which this response occurs indicates that most worms are either prevented from penetrating the intestinal epithelium or are rejected immediately after cell entry. It is proposed that interference with larval infectivity is due to alterations in the epithelial cell apical or brush border membrane. Alterations may result from prior infection or may reflect an acute change induced by challenge infection. In either case the establishment of normal populations of larvae in the mucosa is disturbed. Lectin binding capacity of brush border membranes was used to assess possible membrane alterations. This parameter in uninfected (control) rats was compared with that in infected rats, which acquire resistance to subsequent challenge, and in infected rats immediately after a challenge inoculum. Enriched brush border membrane preparations were characterized for their binding of wheat germ agglutinin, which attaches specifically to the carbohydrate, N-acetylglucosamine. Maximum specific binding of 125I-labeled wheat germ agglutinin occurred within 20 min. The spontaneous rate of dissociation was negligible for 90 min. Highest specific binding resulted at 24 degrees C, pH 6.0 and with 75 micrograms brush border membrane protein per assay tube. Results suggested the existence of multiple binding sites. 1 mg of membrane protein from uninfected rats and rats immunized by primary infection maximally bound 9.8 X 10(10) and 4.3 X 10(10) molecules of wheat germ agglutinin, respectively. Binding for the 'immune' brush border membrane, as compared with the 'uninfected' brush border membrane was reduced during the first 3 weeks of infection and remained low for at least 3 months. No further reduction in binding was observed for brush border membrane isolated within minutes after a secondary infection. These results reveal the induction by a primary infection of changes in brush border membrane structure and the persistence of these changes in the immune host. In view of the rapid turnover time of epithelial tissue the mechanism by which this change is perpetuated speculatively involves immune elements in the lamina propria.     

Accelerated viraemia in cats vaccinated with fixed autologous FIV-infected cells

We have vaccinated cats with fixed autologous FIV infected PBMC to determine whether autologous presentation of antigen is capable of inducing a protective immune response against homologous challenge. To this end autologous PBMC were infected with a FIV molecular clone (19k1). When infection was established, cells were inactivated by dialysis against paraformaldehyde. Upon vaccination, cats developed a virus specific immune response as measured by ELISA against the Gag protein of FIV. No antibodies against the envelope protein were detected with a peptide ELISA. Virus neutralizing antibodies however could be detected with a neutralization assay based on infection of CrFK cells, but not in an assay based on infection of primary T-cells. Although vaccination led to the induction of these virus-specific immune responses, vaccinated cats were not protected against homologous challenge but showed an accelerated viraemia upon infection. This was shown both by PCR and cell-associated viral load. The possible mechanisms underlying this observation are discussed in this paper.     

FIV vaccine studies. II. Clinical findings, hematological changes and kinetics of blood lymphocyte subsets

Five groups of cats were vaccinated with different recombinant feline immunodeficiency virus (FIV) SU vaccines expressed either in Escherichia coli or in the Baculovirus system. In Part I of this series, we described the humoral immune response and outcome of intraperitoneal FIV challenge exposure. Additionally, all cats were monitored for clinical and hematological changes and the course of blood lymphocyte subsets. These results are described in this present paper. A great increase of antibodies was found after vaccination with different recombinant FIV antigens, which did not protect the cats from intraperitoneal FIV challenge infection. This observation was paralleled by an increase of eosinophils during vaccination which was even more pronounced after challenge infection. After FIV challenge, infection lymphadenopathy, gingivitis, pharyngitis, changes in total leukocytes and neutrophils and a decrease in the CD4+:CD8+ ratio were found in cats of all groups and were considered as a sign of the FIV infection taking place, independent of vaccination. The following observations suggest that in these cats a TH2-like immune response was elicited: the high counts of eosinophils, the nature of antigen and adjuvant (aluminium hydroxide) and the high amounts of antigens used for immunization. Clearly, this type of immune response did not protect the animals from intraperitoneal FIV challenge infection.     

Kinetics of immunological responses, resistance to reinfection, and pathological reactions to infection with Trichinella spiralis

Changes in immune responses, resistance to reinfection, and pathological reactions were studied serially in mice that had been infected for four to 40 weeks with 150 larvae of Trichinella spiralis. Immediate footpad hypersensitivity reactions to antigens of Trichinella were present throughout the period of observation. Delayed hypersensitivity reactions 48 hr after injection of antigen were first seen in mice infected for 14 weeks and gradually increased in size thereafter. Intestinal adult worm burdens were determined one week after challenge with 5000 larvae. There was resistance to reinfection and accelerated expulsion of worms in animals challenged three weeks after the primary infection; this resistance waned at seven and 13 weeks but reappeared in mice infected for 20 weeks or longer. Counts of larvae in muscle were determined four weeks after challenge with 5000 larvae. Marked resistance was present four weeks after the primary infection and was maintained for the duration of the study.     

Strongyloides stercoralis: maintenance of exceedingly chronic infections

Two hypotheses were tested to identify the mechanism(s) by which chronic Strongyloides stercoralis infections are maintained in experimental dogs as a model to explain delayed onset recrudescence in humans. Investigations tested the hypotheses that chronic infections result from 1) periodic reactivation of third-stage larvae from a reservoir of dormant parasites outside the gastrointestinal tract or 2) the periodic rejuvenation of postreproductive female worms remaining from a previous infection, lodged in the mucosal crypts. Populations of parenteral larvae survived in mature experimentally infected female dogs for 66 days; individual worms survived for 88 days, but there was no evidence that these larvae re-established patent, adult worm infections. Late in these infections, female worms were present in greater than predicted numbers with no evidence that autoinfection had occurred, suggesting that some postreproductive worms were long-lived. In separate trials, long-lived spent females were once again capable of producing viable larvae when the host was treated with corticosteroids.     

Selective breeding of rats for high and low motor activity in a swim test: toward a new animal model of depression

In three successive experiments, the immune functions of pigs persistently infected with the porcine reproductive and respiratory syndrome virus (PRRSV) have been evaluated. Non-specific immune responses were analyzed over a period of 12 weeks post-infection (PI). In addition, the capacity of PRRSV-infected pigs to develop an efficient immune response against pseudorabies virus (PRV) glycoproteins and to resist to a subsequent virulent challenge was investigated. Our results demonstrate that PRRSV produced minor effects on the immune system of pigs. The skin delayed type hypersensitivity (DTH) in response to phytohemagglutinine injection was slightly diminished one week after challenge, but was restored thereafter. However, three weeks after the infection, the total white blood cell count, and the number of CD2+, CD8+ and IgM+ cells were enhanced. The increase in numbers of CD8+ cells persisted for three consecutive weeks. Serum immunoglobulins in infected pigs also increased by week 3 PI and up to 8 weeks PI. These results show that PRRSV may have stimulating effects on the pig immune system during the phase of long-lasting infection. After immunization with PRV glycoproteins, the production of anti-PRV antibodies and skin DTH response against PRV glycoproteins were not affected. On the contrary, following a virulent PRV challenge, PRRSV-infected pigs developed a better secondary antibody response and their resistance to the infection was as effective as in control pigs. Taken together, our data do not support a systemic immunosuppressive effect of PRRSV, during the persistent phase of infection. Other mechanisms may therefore apply to explain the emergence of secondary infections in endemically infected herds.     

Immunity to Trichinella spiralis. VII. Resistance stimulated by the parenteral stages of the infection

In three experiments mice were given three intravenous injections of Trichinella spiralis newborn larvae produced in vitro. Following a challenge infection of newborn larvae the mice were shown to be on the average 85% resistant i.e., only 15% of the challenge larvae developed to encysted muscle stage larvae in these mice; when the challenge consisted of normal muscle larvae administered per os, the mice were 51% resistant. Neither the number nor longevity of adult worms in the intestines was affected by this immunization procedure.     

Intestinal strongyloidiasis manifesting as eosinophilic pleural effusion

Strongyloidiasis caused by the helminth Strongyloides stercoralis is usually asymptomatic or causes mild dermatologic or gastrointestinal symptoms. In immunocompromised hosts, hyperinfection and dissemination can occur. In this case, I describe a nonimmunocompromised Southeast Asian man who had an eosinophilic pleural effusion, peripheral eosinophilia, and rhabditiform larvae of S stercoralis in the stools. There was complete resolution of the pleural effusion after thiabendazole therapy, thus suggesting strongyloidiasis as the cause of the effusion. Helminthic infections like strongyloidiasis should be considered in the differential diagnosis of an eosinophilic pleural effusion, especially in individuals from endemic areas.     

Stimuli for acquired resistance to Heligmosomoides polygyrus from intestinal tissue resident L3 and L4 larvae

L3 and L4 stages of H.polygyrus were prevented from developing further and were probably killed within 24 h of treatment with ivermectin although total parasite burdens, particularly when treatment was given 4-6 days after infection, declined over a longer period lasting several days. Strong resistance to challenge infection was expressed by infected mice dosed with ivermectin during the tissue phase of larval development. Even immunizing infections as brief as 12-36 h (when only L3 larvae would have been present in the mucosa) elicited strong acquired immunity. When infections were terminated 4-6 days after infection, acquired resistance was 95-100%. The stronger resistance of mice exposed to both L3 and L4 stages was associated with the recognition of low molecular weight polypeptides in adult worm homogenate and there was a highly significant correlation between percentage protection and anti-L4/anti-adult worm serum IgG1 antibodies.     

Expression of glutamate receptor subunit mRNAs in gonadotropin-releasing hormone neurons during the sexual maturation of the female rat

Strongyloides stercoralis (SS) is endemic in tropical and subtropical areas worldwide and in the southeastern United States. The lifecycle of SS is both unique and complex. Human infection begins with the penetration of skin by filariform larvae that migrate hematogenously to the lungs. Larvae then ascend the airway, are swallowed, and mature in the gut. Unlike other nematodes, SS can autoinfect the same host and persist for decades. Categorization of infection includes acute, chronic-uncomplicated, and disseminated forms. Clinical manifestations depend on the particular organs involved. Fifteen to thirty percent of chronically infected people may be asymptomatic. On the other hand, SS may cause the adult respiratory distress syndrome, septic shock, and death. The diagnosis of SS infection is suspected in patients from endemic areas who have blood eosinophilia, and gastrointestinal or pulmonary symptoms. A definitive diagnosis is established by demonstration of SS larvae in stool, body fluids, or tissues. A presumptive diagnosis of SS infection can be achieved by serology. Thiabendazole is the mainstay of treatment, but repeat doses may be necessary if the parasite is not initially eradicated. The low incidence of disseminated SS in areas endemic for both SS and AIDS is surprising and unexplained.     

Cryo-microtome sections of coproculture larvae of Strongyloides stercoralis and Strongyloides ratti as antigen sources for the immunodiagnosis of human strongyloidiasis

Cryo-microtome sections of larvae of Strongyloides stercoralis and S. ratti respectively obtained from human and rat feces cultures, were used as antigens. Fluoresceinate conjugates against human IgG were employed at the ideal titer of 10 for S. stercoralis and 100 for S. ratti. The sensitivity of the indirect immunofluorescence reaction (IIF) was 94.4% and 92.5% and the specificity 94.2% and 97.1% for the two specific larval antigens, respectively. Sera from 123 persons (54 from carriers of S. stercoralis infections and 69 from controls) were submitted to the reaction. The titers of different sera varied from 20 to 2560. There was a significant linear correlation (r = 0.85 p < or = 0.001) between the antibodies from the two species of larval antigens. We conclude that both antigens may be used in the IIF reaction for the diagnosis of human strongyloidiasis. Due to the feasibility of safe and low-cost mass production of S. ratti larvae in the laboratory with a considerable economy of conjugate, their utilization in the serum diagnosis of human strongyloidiasis is recommended.     

Local immune responses to Chlamydia pneumoniae in the lungs of BALB/c mice during primary infection and reinfection

Cell-mediated immune (CMI) responses play a major role in protection as well as pathogenesis of many intracellular bacterial infections. In this study, we evaluated the infection kinetics and assessed histologically the lymphoid reactions and local, in vitro-restimulated CMI responses in lungs of BALB/c mice, during both primary infection and reinfection with Chlamydia pneumoniae. The primary challenge resulted in a self-restricted infection with elimination of culturable bacteria by day 27 after challenge. A mild lymphoid reaction characterized the pathology in the lungs. In vitro CMI responses consisted of a weak proliferative response and no secretion of gamma interferon (IFN-gamma). The number of lung-derived mononuclear cells increased substantially during the primary infection; the largest relative increase was observed in B cells (B220(+)). After reinfection, the number of lung-derived mononuclear cells increased further, and the response consisted mainly of T cells. The reinfection was characterized in vivo by significant protection from infection (fewer cultivable bacteria in the lungs for a shorter period of time) but increased local lymphoid reaction at the infection site. In vitro, as opposed to the response in naive mice, acquired immunity was characterized by a strongly Th1-biased (IFN-gamma) CMI response. These results suggest that repeated infections with C. pneumoniae may induce Th1-type responses with similar associated tissue reactions, as shown in C. trachomatis infection models.     

Mucosal immunity to herpes simplex virus type 2 infection in the mouse vagina is impaired by in vivo depletion of T lymphocytes

Intravaginal (IVAG) inoculation of wild-type herpes simplex virus type 2 (HSV-2) in mice causes epithelial infection followed by lethal neurological illness, while IVAG inoculation of attenuated HSV-2 causes epithelial infection followed by development of protective immunity against subsequent IVAG challenge with wild-type virus. The role of T cells in this immunity was studied by in vivo depletion of these cells with monoclonal antibodies. Three groups of mice were used for each experiment: nonimmune/challenged mice, immune/challenged mice, and immune depleted mice [immune mice depleted of a T-cell subset(s) shortly before challenge with HSV-2]. Mice were assessed for epithelial infection 24 h after challenge, virus protein in the vaginal lumen 3 days after challenge, and neurological illness 8 to 14 days after challenge. Monoclonal antibodies to CD4, CD8, or Thy-1 markedly reduced T cells in blood, spleen, and vagina, but major histocompatibility complex class II antigens were still partially upregulated in the vaginal epithelium after virus challenge, indicating that virus-specific memory T-cell function was not entirely eliminated from the vagina. Nevertheless, immune mice depleted of CD4+ and CD8+ T cells, Thy-1+ T cells, or CD8+ T cells alone had greater viral infection in the vaginal epithelium than nondepleted immune mice, indicating that T cells contribute to immunity against vaginal HSV-2 infection. All immune depleted mice retained substantial immunity to epithelial infection and were immune to neurological illness, suggesting that other immune mechanisms such as virus-specific antibody may also contribute to immunity.