Experimental murine chromomycosis mimicking chronic progressive human disease

Congenitally athymic (nu/nu) mice, mice defective in NK cell and macrophage function (bg/bg), and normal BALB/c mice were inoculated sc with 10 conidia of Fonsecaea pedrosoi (FP). In immunologically intact and immunodeficient mice, a local infection developed approximately 2 weeks post-inoculation and enlarged over 1-2 weeks. In bg/bg and normal nu/+ mice, lesions resolved within 5-6 weeks. However, nu/nu mice continued to have enlarging sc lesions during greater than 4-6 months of observation. These eventually metastasized. Lesions contained few hyphal elements and massive numbers of sclerotic bodies. Five weeks after inoculation, 10 conidia forming units/gm of tissue were recovered from lesions. Delayed type hypersensitivity and serum antibody to FP antigens were demonstrated. Adoptive transfer of lymphocytes from nu/+ mice was followed in 2 months by the resolution of the lesions.     

Detection of histoplasmal antigens in mice undergoing experimental pulmonary histoplasmosis

A micro-ELISA assay was developed for the quantitation of Histoplasma capsulatum antigen in lungs, bronchoalveolar lavage fluid (BALF), and serum of intranasally infected mice. As little as 0.2 ng of antigen/ml could be detected. During the course of experimental histoplasmosis, immunologically intact, thymus-containing mice (nu/+) had detectable histoplasmal antigens in their lungs, serum, and BALF within 1 day of challenge. Lung, BALF, and serum antigen concentration rose to a peak 2 wk after challenge; in nu/+ mice, antigen concentration then declined through the next 2 wk. In contrast, athymic nude mice have depressed cell-mediated immunity; their antigen concentration continued to rise throughout the course of progressive, ultimately lethal, illness. Antigen concentrations correlated with quantitative cultures of the lungs and BALF. There was little cross reactivity in mice challenged intranasally with Candida albicans or Blastomyces dermatitidis. The sensitivity of this test, and the apparently minimal cross reactivity, suggest that the micro-ELISA for histoplasmal antigen might have significant clinical application in diagnosing and monitoring the course of histoplasmosis.     

Cellular immunity to the mouse pneumonitis agent

During infection with the mouse pneumonitis biovar of Chlamydia trachomatis, heterozygous (nu/+) mice with relatively intact T cell function develop both delayed hypersensitivity to C trachomatis antigen and antigen-specific lymphocyte transformation, whereas athymic nude (nu/nu) mice do not. Nu/nu mice are protected against death from mouse pneumonitis by transfer of immune T cells from nu/+ mice, which are more resistant to C trachomatis. While this enables athymic mice to make antibody to C trachomatis (which does not occur without reconstitution), resistance correlates best with development of antigen-specific lymphocyte transformation in the recipient animals. During infection nu/+ mice develop activated alveolar macrophages (by both morphological and functional criteria) while nu/nu mice do not. Nu/+ mice that have been preinfected with Histoplasma capsulatum to activate cellular immunity become more resistant to C trachomatis than do nu/+ controls. Cell-mediated immunity to C trachomatis pneumonia is T cell dependent and is important in host defense.     

Eosinophilia, granuloma formation and migratory behaviour of larvae in the congenitally athymic mouse infected with Toxocara canis

Peripheral blood eosinophilia, histology of skeletal muscle and brain, and larval recovery were compared between congenitally athymic nude mice (nu/nu) and thymus-bearing heterozygous littermates (nu/+) for 6 weeks following oral infection with Toxocara canis eggs. By comparing patterns of peripheral blood eosinophil levels in nu/+ and nu/nu, two types of eosinophilias, one T cell dependent and the other independent, were observed. Eosinophil infiltration and granuloma formation around larvae in the skeletal muscle were weaker in degree in nu/nu than nu/+. The total number of larvae in nu/+ decreased from 2 to 6 weeks after infection. This decrease was directly related to a decrease in larval number in skeletal muscle, not in brain or other tissues. In contrast, no significant decrease of the total number of larvae was observed in nu/nu. The results indicate that eosinophilia, granuloma formation and larval recovery are closely related to cell-mediated immune mechanisms in T. canis -infected mice.     

In vivo significance of T cells in the development of Coxsackievirus B3 myocarditis in mice. Immature but antigen-specific T cells aggravate cardiac injury.

Acute myocardial damage similar to that seen in human myocarditis occurs in BALB/c mice after infection with coxsackievirus B3 (CB3) or encephalomyocarditis virus (EMC). To investigate the role of antigen-specific T cells in the pathogenesis of this disorder, we compared CB3 disease expression in T cell-deficient, athymic nude (nu/nu) mice, in heterozygote (nu/+) mice with normal T cell function, and in nu/nu mice reconstituted with spleen cells from CB3- or EMC-infected nu/+ mice. Acute myocarditis occurred in both nu/nu and nu/+ mice. Severe myocarditis, however, developed only in nu/+ and nu/nu mice reconstituted with CB3-sensitized T cells, but not in those reconstituted with EMC-sensitized T cells. Myocardial virus titer and serum anti-CB3 antibody production were similar in nu/+ and nu/nu groups. Additionally, the presence of Thy 1.2 (pan T), Ly 1 (precursor of other T cell subsets), and Ly 2 (suppressor/cytotoxic T) positive cells was demonstrated in the myocardium in nu/+ and nu/nu mice reconstituted with CB3-sensitized T cells, but not with T cells sensitized by another virus, EMC. These results indicate that immature but antigen-specific T cells play a role in the pathogenesis of ongoing myocarditis.     

Murine amebiasis: the role of the macrophage in host defense

The congenitally athymic nude (nu/nu) mouse was studied as a model for amebic liver disease, using Entamoeba histolytica. Despite intrahepatic inoculation of massive numbers of amebae, we could not produce sustained infections in nu/nu mice. We also failed to induce hepatic amebiasis in thymus-intact nu/+ and +/+ littermates, or in nu/+ mice pretreated with rabbit antimouse thymocyte globulin. Humoral response was measured in both the nu/nu and nu/+ mice. Nu/+ but not nu/nu animals developed a specific IgG response after challenge with E. histolytica. There were no significant IgM responses. Pretreatment with silica increased susceptibility of both nu/nu and nu/+ mice to development of liver abscesses. These studies suggest that host resistance in murine amebiasis is critically dependent upon the macrophage, but not upon T cell-mediated defenses.     

Chronic murine pulmonary blastomycosis induced by intratracheally inoculated Blastomyces dermatitidis conidia

Groups of 6-wk-old male BALB/cByJ mice were injected intratracheally (IT) with viable conidia from Blastomyces dermatitidis strains (FW or CR) harvested from 2-wk-old cultures (Pine-Drouhet agar at 30 degrees C) and separated from hyphal elements by polycarbonate filtration (5 micron). Inocula (0.05 ml) were 93 to 97% conidia in nonpyrogenic normal saline (NPNS) with greater than 90% viability. Quantitative cultures of the lungs and trachea of mice killed immediately after injection of 10(4) conidia (FW) confirmed that the inoculum was evenly distributed between the lungs, with virtually no conidia retained in the trachea. Animals were observed for 354 days for weight change, mortality, extrapulmonary dissemination and histopathologic changes. Mice inoculated with 10(4) FW began dying on Day 44, with a median survival of 92 days. A decrease in mean weight compared with that in control mice was noted by Day 55. In contrast, mice inoculated with 10(4) CR or NPNS neither died with blastomycosis nor lost weight in 329 days. Variation of the FW inoculum by tenfold increments produced comparable dose-dependent alterations in both mortality rate and weight change curves. Fifty percent survival was 32, 36, 92, or 172 days for mice inoculated with 10(6), 10(5), 10(4), or 10(3) conidia, respectively. Dissemination from the lungs to the liver, spleen, kidney, testis, and brain was found in the cachectic mice that were killed. Pathologic features of pulmonary and disseminated infections were comparable to those in human disease. This quantitative, reproducible model of chronic murine blastomycosis mimics the human disease in many respects and should provide a basis for future studies of the immunology, pathogenesis, and therapy of chronic blastomycosis.     

Mammary growth and function and pituitary prolactin secretion in female nude mice

Mammary structural growth in the wholemount preparation, content and synthesis of mammary DNA and RNA estimated by the incorporation of [3H]thymidine and [14C]uridine, pituitary and plasma levels of prolactin and weights and histological structures of some organs of female nude mice (nu/nu) were compared to those of the control (nu/+) with the same genetical background (BALB/c). Both at 3 months of age and on day 1 of lactation, the weights per 100 g body weight of adrenals, spleen and liver of nu/nu mice were significantly higher than those of nu/+ mice. Mammary growth stimulation by pituitary graft was much more marked in nu/nu mice than in nu/+ mice. Slight differences between groups were found in the pituitary and plasma levels of prolactin, in the histological structures of ovaries as well as of the adrenals and thyroids and in the pattern of oestrous cycles. On the other hand, the content and synthesis of mammary DNA at 3 months of age and content and synthesis of both DNA and RNA and RNA/DNA ratio on day 1 of lactation were significantly higher in nu/+mice than in nu/nu mice. All findings suggest that thymus deficient immunosuppression has deleterious effects on mammary growth and function without its alteration in the secretion of prolactin and oestrogen and probably through its decrease in mammary responsiveness to mammotrophins.     

Mechanisms of eosinophilia in Toxocara canis infected mice: In vitro production of interleukin 5 by lung cells of both normal and congenitally athymic nude mice

Mechanisms of eosinophilia were compared between in vitro bone marrow cell cultures of congenitally athymic (nu/nu) mice and their heterozygous littermates (nu/+). Cultures of 5 × 10⁴ bone marrow cells using interleukin 3 (IL-3), IL-5 and granulocyte-macrophage colony-stimulating factor showed that nu/nu and nu/+ mice mimicked each other in eosinophil production both before and after infection with Toxocara canis. Eosinophil differentiating activity (EDA) was detected in media conditioned by spleen cells and lungs of T. canis infected nu/+ mice, although nu/nu mice showed EDA only in lung-conditioned medium. EDA, detected both in infected nu/nu and nu/+ mice, was inhibited by an anti-IL-5 monoclonal antibody. These results indicate that IL-5 may be produced by lung cells of both nu/nu and nu/+ mice as well as by spleen cells of nu/+ mice infected with T. canis, which is the reason why nu/nu mice infected with T. canis exhibit blood eosinophilia.     

Susceptibility of "et," the spontaneously mutating CD1-derived nude mouse, to infection of M. lepraemurium.

We have studied the susceptibility to infection by Mycobacterium lepraemurium (MLM) of a nude, hypothymic, CD1-derived, spontaneous mouse mutant called "et" because of its extraterrestrial appearance. We found that despite their hypothymia, et/et mice were not more susceptible to infection by MLM than their euthymic et/+ counterparts. Infection of both et/et and et/+ mice with 50 x 10(6) bacilli by the intraperitoneal route led only to a mild infection with low levels of antimycobacterial antibodies and a small number of lesions. These lesions were indicative of reactive hepatitis and hyaline perisplenitis with lymphoid hyperplasia. Some small bacilliferous granulomas were also observed at the end of the experiment (5 months of infection). CD1 mice behave in a rather "resistant" manner to the infection by MLM. It is clear that the nu gene is not necessarily linked to the thymus defect, and it is also clear that the hypothymia of et/et mice does not obviously affect their general cell-mediated immune competence.     

Trypanosoma brucei infection in nude mice: B lymphocyte function is suppressed in the absence of T lymphocytes

B lymphocyte function was assessed in outbred nude mice and nu/+controls infected with Trypanosoma brucei brucei. On day 10 of the infection in outbred nu/nu mice in which the initial wave of parasites was strongly controlled, B cell function was unaltered on enhanced compared with uninfected animals or infected nu/+. In other nu/nu mice unable to control the initial parasitaemia, thymidine incorporation and Ig secretion by spleen cells were increased on day 10 and their response to lipopolysaccharide in vitro negated. By day 15 however, even the spleen cells of infected nu/nu which controlled the initial wave of parasites were proliferating and secreting Ig on removal from the mice and they were unable to respond to LPS in vitro. These experiments confirm results of a previous study of B cell function in T cell-depleted mice (Askonas et al. 1979). T. b. brucei infection of mice causes both enhanced Ig production and suppression of the ability of B cells to respond to mitogen even in the absence of T cells, but the presence of T cells may accelerate the changes which occur in B lymphocytes following this infection.     

Immunohistopathology of murine pulmonary histoplasmosis during normal and hypersensitive conditions.

Pulmonary histoplasmosis was induced in nonimmunized and immunized Balb-C nu/+ mice. The lung tissue burden of H. capsulatum, histopathology, the size of the inflammatory area, and the numbers of total T lymphocytes and subtypes in situ were evaluated serially after challenge. Over 3 days previously immunized mice developed a large lymphocyte/macrophage inflammatory response. This rapidly decreased in the next 2 wk. In contrast, the nonimmunized control mice developed a predominantly polymorphonuclear infiltrate that evolved more slowly over the first week of infection. This initial response was nonspecific but, after the first week, shifted to lymphocytes and granuloma formation. The lymphocyte infiltration in both immunized and nonimmunized mice was predominantly CD4. Previously immunized mice had a rapid decrease in tissue counts of H. capsulatum after Day 3, but nonimmunized mice continued to have increased counts through Day 7 after infection. These studies reproduce in the mouse model a host response similar to acute reinfection histoplasmosis in the human. In this condition an intense cell-mediated inflammatory response is thought to be elicited by fungal antigens and to represent host reaction rather than fungal replication. Our experimental model added new information relevant to the understanding of the pathogenesis of this process.     

Antigenic variation in Giardia lamblia: infection of congenially athymic nude and scid mice

Athymic nude mice of the outbred Zur:ICR-nu and inbred BALB/c strain and scid mice were infected with a cloned human isolate of Giardia lamblia (GS/M-83-H7). Changes in the expression of the major surface epitope of the intestinal trophozoites (characterized by the binding capacity of monoclonal antibody MoAbG10/4) as well as cellular and humoral immune parameters of the hosts were followed during the course of infection. Self-cure was observed in heterozygous (nu/+) BALB/c mice by day 22 post-infection (p.i.) and in heterozygous (nu/+) Zur:ICR-nu strain by day 65 p.i. Homozygous (nu/nu) mice of both strains remained chronically infected until end of the experiments (day 45 p.i. for BALB/c mice and day 122 p.i. for Zur:ICR-nu mice, respectively). Only heterozygous (nu/+) mice were able to mount a gut-associated (Peyer's patch) lymphoproliferative response to G. lamblia antigen. Therefore, T-cell dependent mechanisms were necessary for a self-cure. Antigenic variation occurred in all nu/+ and nu/nu animals of both strains. Trophozoites expressing the major surface epitope (assessed by direct immunofluorescence with FITC-labelled MoAb G10/4) decreased to zero by day 22 p.i. In contrast, the proportion of trophozoites expressing the major surface epitope in infected scid mice remained at the initial level (greater than 99%) until termination of the experiment (day 25 p.i.); therefore, antigenic variation did not occur. All nu/nu and nu/+ mice but not scid mice demonstrated a humoral immune response to G. lamblia antigen. These experiments suggest functional B-cell dependent mechanisms are most likely responsible for the surface antigen switch. Transfer of infection occurred naturally from experimentally infected scid-mice to their mother, proving the initial antigenic surface variant remains unchanged after encystment and subsequent excystment followed by infection in a new host.     

T cell-mediated immune response enhances the severity of myocarditis in secondary cardiotropic virus infection in mice

In this report, we showed that a previous enterovirus exposure in ordinary mice with normal T cell function, but not in T cell-deficient mice, can influence development of myocardial inflammation with a second virus exposure. Inoculation of 4-week-old male BALB/c-nu/+ (euthymic and normal T cell function) mice with amyocarditic Coxsackie virus B1 (CB1), followed by inoculation 28 days later with myocarditic variant of Coxsackie virus B3 (CB3-m) resulted in more intense myocardial inflammation and injury than was seen in BALB/c-nu/+ inoculated with CB1, followed by inoculation with non-enterovirus, i.e., encephalomyocarditis virus (EMC) or influenza A virus and in age-matched BALB/c-nu/+ mice secondary inoculated with CB3-m alone. In contrast, this phenomenon of the enhancement of the severity of myocarditis by a secondary CB3-m inoculation was not seen in BALB/c-nu/nu (athymic and T cell- deficient) mice. Interestingly, inoculation of BALB/c-nu/+ mice with CB1, followed by inoculation 28 days later with another amyocarditic variant of Coxsackie virus B3 (CB3-o), resulted in more severe myocarditis than was seen in age-matched BALB/c-nu/+ mice secondary inoculated CB3-o alone. Myocardial-activated T cells and elevated serum interleukin-6 were involved in the exacerbation of the disease during the reinfection. T cell-mediated immune responses to a conserved antigenic epitope among the enteroviruses may be involved in the exacerbation of myocardial inflammatory disease during a second enterovirus infection. Received: 14 December 2000, Returned for revision: 19 January 2001, Revision received: 30 January 2001, Accepted: 31 January 2001     

Immunity to Brugia pahangi in athymic nude and normal mice: eosinophilia, antibody and hypersensitivity responses

Congenitally athymic nude (nu/nu) mice, immunologically reconstituted by thymus grafting before inoculation with infective larvae, and mice heterozygous for the nu gene (nu/+), mounted potent protective humoral and cellular immune responses to Brugia pahangi. Although responses were not identical, both groups of mice produced IgM, IgG and IgE antibodies specific for adult worm antigen (S-Ag) present in a crude aqueous extract, made immediate and delayed hypersensitivity footpad swelling responses when challenged with S-Ag and eliminated their infection in the early larval stages. Heterozygotes also exhibited a marked eosinophilia which peaked coincident with larval killing. In contrast, thymus grafting of patent nudes had no effect upon microfilaraemias or adult worm burdens and did not completely protect against a challenge larval inoculum although antibodies specific for S-Ag were produced. With the occasional exceptions of moderate immediate footpad swelling and very low titres of IgM specific for S-Ag, no specific immune responses to B. pahangi were found in ungrafted nude mice which allowed full development of adult worms and supported patent infections.     

Application of an immunofluorescence assay for Crohn's disease using primed nude mouse lymph nodes to a United States/Mexican border population

Previous studies using an indirect immunofluorescence (IF) technique found that sera from patients with Crohn's disease (CD) frequently contained antibody that recognized an antigen(s) in CD tissue filtrate-primed lymphoid tissue from athymic nude (nu/nu) mice. The present study examined the reproducibility of this IF assay in a different laboratory setting and used patients with infectious diarrhea. We examined the immunoreactivity of 113 sera from eight patients with Crohn's disease, 11 with ulcerative colitis, 62 patients with infectious diarrhea, 22 nondiarrheal disease controls, and ten normal adults. Sera were absorbed with nu/+BALB/c mouse serum proteins coupled to Sepharose 4B to remove nonspecific binding. All sera were coded and tested against at least 2 of 4 lymph nodes from nu/nu mice that were previously inoculated with either CD tissue filtrate or with a passaged CD-induced nu/nu lymphoma. Reference positive CD sera and negative control sera were run in parallel during each assay. Sera from three CD patients consistently showed positive IF. None of the sera from patients with infectious diarrhea, other disease controls, or normals were positive. These results demonstrate the reproducibility of the IF assay using CD tissue-primed nude mouse lymph nodes and the preferential immunoreactivity of CD sera.     

Mouse model of pulmonary blastomycosis: utility, simplicity, and quantitative parameters

A murine model of pulmonary mycotic disease using Blastomyces dermatitidis is defined quantitatively. Intranasal inoculation with yeasts avoids delays in growing microconidia and can be performed with routine precautions without sophisticated biohazard facilities. The particles rapidly reach the bronchioles and alveoli, producing a progressive focal pneumonia, studied histologically. In vitro growth conditions are defined to titer inocula reproducibly with visual methods. Median survival can be titrated with the challenge inocula. Although initial postchallenge lung cultures are not always positive, replication eventually proceeds, at a rate defined for various challenge sizes, to a ceiling of 10(7) to 10(8) colony-forming units per g of tissue incompatible with life. Lung weight correlates closely with colony-forming units per g above a threshold. Lethality correlates with initial body weight and with age. During the course of infection, weight is an accurate predictor of mortality. Extrapulmonary dissemination occurs as a premortem event in protracted infections. A wide variation in fungal strain virulence is documented. Quantification of these features of the model makes it useful for study of host response or therapeutic intervention, because efficacy can be related to stage of disease and parasitic burden.     

Treatment of murine cryptococcosis with cyclosporin-A in normal and athymic mice

We previously demonstrated that prophylactic Cyclosporin-A (Cs-A) treatment of mice enhanced survival after inoculation of Cryptococcus neoformans by both the intratracheal (IT) and intravenous (IV) routes. In the present studies, we determined whether an established infection due to C. neoformans could be treated with Cs-A. Mice inoculated IT develop a prominent pulmonary infection with late dissemination to distant organs. The survival of mice infected by the pulmonary route that received Cs-A subcutaneously was prolonged in both immunologically intact and congenitally T-cell-deficient mice (athymic nude mice). In normal mice that received Cs-A, growth of C. neoformans was arrested in the lung, spleen, kidney, and liver, and the rapid rate of accumulation of organisms in the brain was slowed as compared to that of control mice. In nude mice, the organisms continued to increase in all organs although at a considerably slower rate than in untreated control nude mice. Mice given C. neoformans IV developed infection in the brain at the time of inoculation. When an inoculum was deposited in the brains of normal mice by giving the organism IV and Cs-A treatment initiated 3 days later, mice receiving Cs-A did not demonstrate a reduced number of C. neoformans in the brain as compared to untreated control mice. Thus, Cs-A was effective for treatment of extraneural cryptococcal infection in normal mice, but it was unable to reduce cryptococcal replication in the central nervous system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ketoconazole inhibition of intracellular multiplication of Trypanosoma cruzi and protection of mice against lethal infection with the organism

The effects of ketoconazole against infection with Trypanosoma cruzi both in vivo and in vitro were examined. In vivo, ketoconazole significantly protected mice infected with lethal inocula of the Y strain of T. cruzi even when treatment was initiated seven days after infection; protection was also demonstrated for three other strains. Although mice had demonstrable parasitemia after completion of therapy, tissue sections of treated mice revealed a complete absence of organisms. Concentrations of ketoconazole as low as 0.001 microgram/ml inhibited in vitro replication of intracellular organisms, whereas concentrations of ketoconazole that prevented replication of intracellular amastigotes had no effect on extracellular organisms. This finding and the observation that inhibition of replication of amastigotes occurred in macrophages exposed to ketoconazole before infection suggests that the inhibitory effect depends on interaction of the drug with host cells. Thus ketoconazole should be tested as a therapeutic agent for Chagas' disease in humans.     

Immunopathology of Schistosoma japonicum infection in athymic mice

Athymic (nu/nu) mice and heterozygous littermate controls (nu/+) were examined 7 and 10 weeks after infection with 10 cercariae of Schistosoma japonicum. Schistosome infection developed normally in both groups of mice and eggs were produced in normal numbers. Nu/nu mice developed small circumoval granulomas with minimal fibrosis while nu/+ mice developed large fibrotic granulomas. Unlike the mononuclear responses to S. mansoni eggs at 7 weeks, those to S. japonicum often were abscess like with narrow rims of liver cell necrosis or microvesicular fatty change. However, evolving granulomas in nu/+ mice were enriched with eosinophils, epithelioid macrophages, immature granulocytes and plasma cells, all scarce in the corresponding nu/nu lesions as were fibroblasts and collagen fibres, thus accounting for their smaller mean size and better healing. Our aggregate evidence shows that normal granuloma formation and cellularity in S. japonicum infection is controlled by T-cells as is the case for S. mansoni, and not by antibodies or immune complexes.