Experimental mucormycosis in congenitally athymic (nude) mice

Athymic nude (nu/nu) mice and their phenotypically normal (nu/+) littermates displayed a similar susceptibility to acute lethal infection withAbsidia corymbifera. Although the clinical manifestations of acute infection were also similar in both groups, the nude mice tended to develop more extensive lesions and were less effective in eliminating viableA. corymbifera spores than their heterozygous littermates. The results suggested that thymus-dependent processes did not play an essential role in primary resistance to mucormycosis.     

Modulation of macrophage Ia-expression by lipopolysaccharide. I. Induction of Ia expression in vivo

Experiments were performed to analyze the modulation of macrophage Ia expression and biosynthesis by Salmonella minnesota-derived lipopolysaccharide (LPS) in vivo. The i.p. injection of LPS into LPS-responder mice caused a dramatic increase in the Ia expression of the peritoneal macrophage population harvested 1 wk after injection. As little as 1 ng of lipid-rich Re595 LPS per mouse caused a significant I-Ak increase, and 1 microgram was optimal; wild-type S. minnesota LPS was less active. No I-Ak induction by LPS was observed in the LPS-nonresponder strain C3H/HeJ. LPS-induced macrophages showed a 6- to 16-fold increase in I-Ak expression by radioimmunoassay (RIA), a 3- to 10-fold increase in the proportion of I-Ak-positive cells, and a 10- to 15-fold increase in I-Ak biosynthetic capacity. The magnitude of this induction by LPS was comparable to increases observed after injection of live Listeria monocytogenes. The kinetics of I-Ak induction by LPS and by L. monocytogenes were different: LPS caused an initial decrease in I-Ak expression 1 day after injection, and I-Ak induction by LPS occurred more slowly and maintained heightened expression longer. Several H-2 gene products (H-2Kk, I-Ak, and I-Ek) were augmented in LPS-induced macrophages. In keeping with increased I-A and I-E expression, LPS-induced macrophages were more effective than normal macrophages in presenting antigen to T lymphocytes. We suggest that the modulation of macrophage Ia expression is one important mechanism contributing to the immunoregulatory activity of LPS.     

In vivo generation of suppressor T cells by thymosin in congenitally athymic nude mice

Treatment of nude mice with thymic factors such as thymosin has been mostly ineffective in generating effector T cells. This study examined the effects of treating nude mice with thymosin fraction 5 on the induction of cells that could participate in and/or regulate cytotoxic T lymphocyte (CTL) generation by normal spleen cells in vitro. Splenic lymphocytes from BALB/c nude mice injected with thymosin fraction 5 every other day for 2 wk were tested for their ability to generate CTL in vitro. Two days after the last subcutaneous injection of thymosin, nude spleens were removed, mixed with normal BALB/c spleen cells, and placed into a mixed lymphocyte tumor culture (MLTC) against allogeneic RBL 5 tumor cells. After a 5-day incubation, cultures were tested for the presence of CTL in a 4-hr 51Cr-release assay. Spleen cells from thymosin-treated nude mice did not generate CTL but suppressed the ability of normal spleen cells to generate CTL in vitro. Characterization of the thymosin-induced nude mouse suppressor cells showed them to be Thy 1 positive, nonadherent, cyclophosphamide-sensitive T cells. These data demonstrate that some T cell maturation occurs in vivo under thymosin influence. However, the activity of these cells is initially limited to a regulatory function. These studies suggest that maturation of functional suppressor T cells occurs before CTL. Further immunologic manipulation appears to be necessary in order to induce CTL effector cells in nude mice.     

Role of bacterial hemolysin production in induction of macrophage Ia expression during infection with Listeria monocytogenes

The production of a hemolytic exotoxin (Hly) termed listeriolysin O (LLO) is a major determinant of the virulence of the Gram-positive bacterium Listeria monocytogenes. As determined by lethal inoculum size, LLO- strains of L. monocytogenes generally are several orders of magnitude less virulent than their LLO+ counterparts. The generation of protective anti-Listeria T cell immunity also has been shown to depend on the LLO phenotype of the bacteria present during primary infection, although the cellular basis of this observation is not known. The experiments described here address the role of LLO in regulation of the expression of class II MHC (Ia) molecules by murine macrophages. Because Ia expression by macrophages and other APC is thought to be a central factor in the generation of T cells specific for bacterial Ag, we have tested the hypothesis that the failure of LLO- strains to elicit anti-Listeria T cell responses might be secondary to an inability of these strains to stimulate increases in macrophage Ia levels. Our results show that the macrophage Ia response after i.p. injection of L. monocytogenes correlates strongly with the LLO phenotype of the bacteria. The presence of LLO+ organisms, even at very small numbers (as few as 10), elicits a striking increase in Ia expression by peritoneal macrophages. In contrast, even at very high numbers (up to 10(6) per mouse), LLO- bacteria fail to stimulate a strong Ia response. We also have analyzed macrophage Ia expression after injection of lysates of Escherichia coli expressing recombinant LLO protein. Similar to the results obtained with LLO+ and LLO- L. monocytogenes, we have observed Ia induction only with LLO+ lysates. Ia induction by this crude recombinant LLO preparation can be inhibited by cholesterol or heat. Furthermore, supernatants derived from cultures of LLO+ (but not LLO-) L. monocytogenes can cause Ia induction when administered via i.p. injection. Taken together, these findings suggest that the failure of macrophages to respond to LLO- organisms with an increase in Ia expression may be a major underlying cause of the failure of these bacteria to induce Listeria-specific protective T cell immunity. Furthermore, we propose that the induction of macrophage Ia expression in response to bacterial toxins such as Hly may represent one component of a set of early, innate immune mechanisms, and that this induction may provide a critical "bridge" to later, acquired, Ag-specific immune processes.     

Induction of macrophage interleukin-1 production by Listeria monocytogenes hemolysin.

Listeriolysin O produced by a hemolytic strain of Listeria monocytogenes was purified from the ammonium sulfate precipitate of a culture supernatant through the steps of ion-exchange chromatography and gel filtration. The purified hemolysin finally gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 58,000. When peritoneal exudate macrophages were stimulated with purified hemolysin, we found a high level of IL-1 activity as determined by thymocyte costimulator assay in the culture supernatant. Cell-associated and intracellular IL-1 activity was also detected. The activity in the supernatant or membrane was blocked by polyclonal antibody to murine IL-1 alpha. Moreover, IL-1-specific mRNA expression could be detected in the macrophages stimulated with listeriolysin O by Northern blot analysis. Possible contamination by LPS of the listeriolysin O preparation did not seem to contribute to the induction of macrophage IL-1 production.     

Immunosuppression of congenitally athymic (nude) mice with heterologous anti-immunoglobulin heavy-chain antisera

Congenitally athymic (nude) mice have been shown to be far more sensitive than their phenotypically normal littermates to immunosuppression by anti-Ig antisera. Anti-μ suppression of nude mice was seen to result in complete and stable loss of IgM and IgA as well as severe reductions in IgG1 and IgG2 levels. Anti-α treatment of nudes resulted only in complete and stable loss of IgA; similarly, anti-γ1γ2 treatment achieved only class-specific reductions of IgG1 and IgG2 levels, but these reductions recovered slightly during suppression. Nude mice were more severely immunosuppressed than their phenotypically normal littermates upon anti-Ig treatment and demonstrated much less ability to recover from such suppressive effects. The significance of these observations regarding thymus dependency of immunoglobulin synthesis in nude mice is discussed.     

Response to Listeria monocytogenes in mice lacking MHC class Ia molecules

MHC class Ia-deficient mice (H2 Kb-/- Db-/-) inoculated with the intracellular pathogen Listeria monocytogenes (LM) displayed a three- to fourfold expansion of splenic CD8+ T cells 6 days following infection. Culture of these spleen cells in vitro gave rise to CTL that recognized LM-infected target cells and were restricted by the class Ib molecules, Qa1b and M3. Exposure of target cells to heat-killed LM (HKLM) rather than live bacteria did not result in CTL-mediated lysis. Target cells pulsed with three LM peptides known to bind M3, f-MIGWII, f-MIVTLF, and f-MIVIL, were recognized by effector cells from both B6 and Kb-/- Db-/- animals. In vivo analysis showed that B6 and Kb-/- Db-/- mice clear LM from the spleen and liver rapidly with similar kinetics, whereas TAP.1-/- mice, which are deficient in class Ia and Ib molecules, clear LM slowly upon infection. To establish the in vivo role of CD8+ T cells in Kb-/- Db-/- animals, we showed that depletion of such cells from the spleens of immune mice prevented the adoptive transfer of protective immunity to syngeneic recipients. Spleen cells from Kb-/- Db-/- mice were also capable of generating responses directed against syngeneic as well as allogeneic class Ia molecules in vitro. Thus, class Ia-deficient animals have a CD8+ T cell repertoire capable of recognizing both class Ia and class Ib molecules and can generate protective immunity to LM.     

Effect of immune reconstitution on resistance to Brugia pahangi in congenitally athymic nude mice

The dichotomy of resistance to Brugia pahangi (Nematoda: Filarioidea) between nonsusceptible, euthymic C3H/HeN mice, heterozygotic for the "nu" gene (+/nu), and susceptible, congenitally-athymic "nude" (nu/nu) C3H/HeN mice, suggests that resistance is thymus-dependent. To test this hypothesis, the effect of syngeneic neonatal thymus grafts and neonatal thymus cell suspensions on recovery of worms at day 40 PI, and responses to Concanavalin A (Con A) were examined in reconstituted nudes. Nude recipients of a thymus graft 7 or 14 wk before subcutaneous inoculation with 50 infective larvae (L3) yielded no worms and responded strongly to Con A. Serum from these mice reacted in two lines of identity with serum from similarly-infected heterozygotes by double radial immunodiffusion against an adult worm saline extract. Nude recipients of a thymus 2 days or 3 wk before inoculation harbored an average of three or two worms, respectively. Intravenous injection of nude recipients with 10(7) or 10(8) neonatal thymus cells seven weeks before inoculation was less effective in conferring resistance to B. pahangi and responsiveness to Con A. Complete resistance to B. pahangi could be adoptively transferred to nude mice by 10(8) spleen cells obtained from infection-primed heterozygotes and injected intravenously on the day of larval inoculation. The same numbers of worms were significantly reduced. less effective when injected 3 wk before inoculation, although numbers of worms were significantly reduced. Passive transfer of primed heterozygote serum, containing high titers of antibodies to adult worm and larval antigens, failed to protect nude recipients against a larval inoculum in the absence of cellular reconstitution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of T-lymphocytes on normal haemopoiesis: studies in congenitally athymic nude mice.

The germ-free nude mouse represents a most useful animal for investigating the effects of a congenital deficiency of T-lymphocytes on various physiological processes, including haemopoiesis. Nude mice (nu/nu) of the CBA strain, nonmutant inbred CBA mice, and inbred C3H mice were reared in germ-free isolators and used to compare (1) the haematological parameters of nu/nu mice and CBA mice at different ages and (2) the labelling pattern of circulating leucocytes at various times after a single intraperitoneal injection of 3H-thymidine into 3-month-old nu/nu and C3H mice. The data suggest that the deficiency of T-lymphocytes in nu/nu mice may lead to a disturbance of haemopoiesis in 2 to 6-month animals which is characterised by a mild macrocytosis and a very marked reduction in the proportion of labelled leucocytes which can be seen in the blood after an injection of 3H-thymidine. Despite these perturbations, unstressed nu/nu mice were able to maintain adequate numbers of blood cells (other than lymphocytes) in their circulation. Nine-month-old nu/nu mice did not show a macrocytosis and the disappearance of the marcrocytosis at this age was associated with an increase both in the blood lymphocyte count and in the mass of lymphoid tissue in the spleen and mesenteric lymph nodes.     

Mycoplasma pulmonis arthritis in congenitally athymic (nude) mice. Clinical and biological features

Congenitally athymic BALB/cA nu/nu mice were employed to elucidate the role of the thymus in experimental Mycoplasma pulmonis strain m53 infection, and nu/+ mice were used for comparison. Chronic polyarthritis was frequently produced in both of nu/nu and nu/+ mice by intravenous injection of the organisms. Macroscopically, nu/nu mice developed severer arthritis and a much lower grade of resolution than nu/+ mice. Periarticular abscess, conjunctivitis, and emaciation were observed in some of the nu/nu mice, but not in the nu/+ mice. Mycoplasmas were isolated from joints and other tissues (including periarticular abscesses and eyelids) of infected nu/nu mice at higher frequencies as well as in greater quantities, and did not show any elimination trends for at least 20 weeks after inoculation. However, nu/+ mice, mycoplasmas were almost exclusively located in joints, and distribution of organisms to the other organs disappeared soon after the infection. Increases in complement-fixing antibody titers were not related to the inhibition of mycoplasmal spread. Thymus-dependent functions that may in some way prevent growth and spread of mycoplasmas in mice are discussed.     

Lack of susceptibility of congenitally athymic nude mice to human non-A, non-B hepatitis

Nude mice were inoculated intravenously with chimpanzee serum containing a human non-A, non-b hepatitis agent. Control groups of nude mice were inoculated with normal saline or normal chimpanzee serum. During 77 days of observation, evidence of non-A, non-B hepatitis was not detected. Serum alanine aminotransferase levels remained within normal limits, and normal liver histology was seen in serially killed mice.     

Hymenolepis nana: worm recovery from congenitally athymic nude and phenotypically normal rats and mice

When eggs or mouse-derived cysticercoids of Hymenolepis nana were inoculated into previously uninfected congenitally athymic nude (rnu/rnu) rats of an outbred Rowett strain, they failed to mature in the intestinal lumen. They also failed to mature in phenotypically normal (rnu/+) littermates, except when these hosts were treated with cortisone acetate from the beginning of the lumen phase. The Rowett rat, either thymus-deficient or not, was susceptible to tissue cysticercoids but resistant to luminal adults. It is therefore considered to be an unnatural host, at least for mouse-derived H. nana. There was little or no difference in susceptibility to initial tissue cysticercoids between these nude rats and phenotypically normal ones. The normal rats became completely resistant to reinfection with eggs and no secondary cysticercoids developed in their intestinal tissue, whereas the nude rats showed unaltered susceptibility to secondary tissue cysticercoids. Thus, acquired resistance to egg challenge, assessed by the failure of tissue cysticercoid recovery, was thymus-dependent. However, innate resistance to both a primary egg dose, assessed by the low recovery rates of tissue cysticercoids, and to a primary cysticercoid dose, assessed by the failure of luminal adult recovery, were thymus-independent. The effect of cortisone acetate to initiate maturation of H. nana appeared to be unrelated to thymus function. In contrast, all mice, either thymus-deficient or not, were highly susceptible to both phases. The number of worms recovered was more than 10 times greater than that of cysticercoids established in the rat's intestinal tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Colonization of congenitally athymic, gnotobiotic mice by Candida albicans

Colony counts, scanning electron microscopy, and light microscopy were used to assess the capacity of Candida albicans to colonize (naturally) and infect the alimentary tract of adult and neonatal (athymic [nu/nu] or heterozygous [+/nu] littermates) germfree BALB/c mice. When exposed to yeast-phase C. albicans, the alimentary tract of adult germfree mice (nu/nu or +/nu) is quickly (within 24 to 48 h) colonized with yeast cells. Neither morbidity nor mortality was evident in any mice that were colonized with a pure culture of C. albicans for 6 months. Yeast cells of C. albicans predominated on mucosal surfaces in the oral cavities and vaginas of adult athymic and heterozygous mice. In both genotypes, C. albicans hyphae were observed in keratinized tissue on the dorsal posterior tongue surface and in the cardial-atrium section of the stomach. Conversely, neonatal athymic or heterozygous mice, born to germfree or C. albicans-colonized mothers, do not become heavily colonized or infected with C. albicans until 11 to 15 days after birth. Although yeast cells adhered to some mucosal surfaces in vivo, neither widespread mucocutaneous candidiasis, i.e., invasion of mucosal surfaces with C. albicans hyphae, nor overwhelming systemic candidiasis was evident in neonatal (nu/nu or +/nu) mice. Thus, even in the absence of functional T-cells and a viable bacterial flora, athymic and heterozygous littermate mice (adult or neonatal BALB/c) that are colonized with a pure culture of C. albicans manifest resistance to extensive mucocutaneous and systemic candidiasis.