Infection of BALB/c mice with the filarial nematode Litomosoides sigmodontis: role of CD4+ T cells in controlling larval development.

Litomosoides sigmodontis is the only filaria which develops from infective larvae into adults in immunocompetent laboratory mice. Depletion of CD4+ T cells from infected BALB/c mice resulted in worm and microfilarial burdens significantly higher than those of infected controls. Th2 cytokines, eosinophilia, and immunoglobulin E, which were strongly induced in infected controls, were diminished in CD4-depleted mice.     

Interleukin-4 is essential for the control of microfilariae in murine infection with the filaria Litomosoides sigmodontis

Litomosoides sigmodontis is the only filaria which develops from infective larvae into microfilaria-producing adults in immunocompetent laboratory mice. In this study we report that interleukin-4 knockout (IL-4 KO) mice have an up to 100-fold-higher and a significantly prolonged microfilaremia compared to wild-type BALB/c mice, as well as 20 times more microfilariae in the thoracic cavity, the site of infection. While worm development and adult worm persistence were equivalent in IL-4 KO and wild-type mice, the fertility and length of adult female worms in IL-4 KO mice was clearly enhanced. The high susceptibility to microfilariae in IL-4 KO mice required the presence of adult worms in a full infection cycle since microfilariae loads did not differ much between IL-4 KO and wild-type mice when purified microfilariae were injected into mice. In addition, we found that eosinophilia was diminished and immunoglobulin E (IgE) was absent in IL-4 KO mice. IgE, however, does not seem to be the essential factor for microfilarial containment since microfilaremia was not elevated in B-cell KO mice. In conclusion, IL-4 is shown for the first time to be essential for the control of microfilarial loads but not of adult worm loads in a fully permissive murine filarial infection. IL-4 dependent effector pathways seem to operate on adult worms rather than directly on microfilariae.     

Developmental patterns of seizure susceptibility in inbred strains of mice.

Samples of mice from each of 6 inbred strains were tested for audiogenic and electroconvulsive seizures at 5 ages (14, 21, 28, 35, and 42 days). A moderately large within-strain correlation (.67) was found, indicating that developmental patterns of susceptibility to audiogenic and electroconvulsive seizures are similar within each strain. The finding of an even larger between-strain correlation (.91) indicated that strains which are highly susceptible to audiogenic seizures are also likely to be highly susceptible to electroconvulsive seizures. In a 2nd experiment, whole brain norepinephrine and serotonin were assayed in each of 5 inbred strains at 21 and 42 days of age. Results were consistent with the hypothesis that levels of these amines are inversely related to seizure susceptibility. Mice from strains which were susceptible to seizures at 21 days of age had significantly lower levels of norepinephrine and serotonin in brain than did 42-day-old, seizure-resistant animals.     

Senile ocular amyloidosis in SAM and BALB/c strains of mice

We investigated whether amyloid deposition can affect retinal atrophy in old SAMR1, SAMP1 and BALB/c mice. Immunohistochemistry revealed that old SAMP1 mice showed the deposition of the murine senile amyloid protein fibril, AApoA-II in the subconjunctival tissue, the vessel walls near the chamber angle, and the sheaths of the external ocular muscles and the conjunctival glands, but was never observed in the retina or the choroid. Although the old SAMR1 mice also showed a remarkable loss of retinal photoreceptor and ganglion cells, they never showed any amyloid deposition. The BALB/c strain did not showed any amyloid deposition either. Our data suggest that atrophy of the retina is not related to senile systemic amyloidosis in mice.     

Microfilariae of the filarial nematode Litomosoides sigmodontis exacerbate the course of lipopolysaccharide-induced sepsis in mice

Helminths facilitate their own survival by actively modulating the immune systems of their hosts. We investigated the impacts that different life cycle stages of the rodent filaria Litomosoides sigmodontis have on the inflammatory responses of mice injected with sublethal doses of lipopolysaccharide (LPS). Mice infected with female adult worms from prepatent infections, worms which have not yet started to release microfilariae, developed lower levels of proinflammatory cytokines in the peripheral blood after LPS challenge than sham-treated controls, demonstrating that female adult worms can mitigate the innate immune response. The presence of microfilariae in mice, however, through either direct injection or implantation of microfilaria-releasing adult female worms, turned the LPS challenge fatal. This lethal outcome was characterized by increased plasma levels of gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), interleukin 12 (IL-12), and IL-6, greater numbers of macrophages and granulocytes in the peripheral blood, and decreased body temperatures in microfilaria-infected mice. Microfilaria-infected mice deficient in IFN-gamma receptor and TNF receptor 1 had increased survival rates after LPS challenge compared to immune-competent mice, suggesting that microfilariae worsen LPS-induced sepsis through actions of IFN-gamma and TNF-alpha. In summary, we have demonstrated that infection of mice with L. sigmodontis female adult worms from prepatent infections protects mice injected with LPS whereas microfilariae worsen LPS-induced sepsis through the induction of proinflammatory cytokines and upregulation of granulocytes, NK cells, and monocytes in the peripheral blood.     

Avoidance conditioning in nine strains of inbred mice using optimal stimulus parameters

A total of 66 mice from nine highly inbred strains were tested on an avoidance conditioning task using optimal stimulus parameters derived from previous research. CS-US interval was set at 3 sec, intertrial interval at 120 sec, and shock at 400 v. These parameters were successful in differentiating strains, yielding a number of significant differences, with SWR/J the fastest-conditioning strain and BALB/cJ the slowest-conditioning strain.This research was supported by National Research Council of Canada Grant APB-105.     

A comparative analysis of radiation- and virus-induced leukemias in BALB/c mice

Endogenous murine leukemia virus (MuLV) proviral copies were analyzed in thymomas induced in normal BALB/c (Fv-1b) and in Fv-1n congenic mice by X-irradiation. Both strains of mice developed leukemia with similar kinetics, indicating that N-tropism of endogenous MuLV was not a rate-limiting factor in development of disease. Southern blot analysis, using a probe specific for ecotropic virus and for ecotropic-specific sequences retained in pathogenic, env-recombinant viruses, showed that the majority of radiation leukemias lacked newly acquired, clonally integrated, proviruses. This was in contrast to virus-induced leukemias, which routinely exhibited several new proviral integration sites. When an internal proviral DNA restriction fragment was monitored, some radiation leukemias showed evidence of nonclonal infection, accounting for more frequent isolation of infectious virus from such leukemias. Differences in expression of T-cell surface antigens were found in X-ray-induced and virus-induced leukemias. All radiation leukemias were TL positive, whereas virus-induced leukemias were primarily negative for TL. Some differences were also found in Lyt-1 and Lyt-2 expression. The data as a whole suggest that, in the majority of cases, radiation leukemogenesis is not initiated by a viral route--that is, the sort of viral mechanism for which exogenous infection by known pathogenic MuLV is the paradigm.     

Experimental production of actinomycetoma in BALB/c mice.

Chronic actinomycetoma associated with grain production was induced in BALB/c mice by subcutaneous inoculation of live Nocardia brasiliensis in Freund incomplete adjuvant into the hind footpads. Similar inoculation of N. asteroides and N. caviae resulted in local tumor formation which healed spontaneously after 5 months, the disease disseminating into the peritoneum, where masses or organisms could be detected. Grains were recovered from superficial skin lesions of N. caviae, but not from the N. asteroides-infected mice. Mycetoma lesions, appearing as early as 1 month after inoculation of 1.2 X 10(7) colony-forming units of N. brasiliensis per ml or as late as 3 months with inoculation of 1.0 X 10(5) colony-forming units per ml, became persistent and were readily detectable even 6 months after inoculation. No spontaneous healing occurred, and grains were recovered at different stages of the disease. Saline suspensions of N. brasiliensis also produced typical mycetoma lesions, although the incubation period was ca. 6 months. Adjuvant addition appeared to accelerate the onset of the disease. Experimental production of actinomycetoma in laboratory animals allows the study of many unanswered aspects of the disease and also provides a suitable model for therapeutic trials in the search for new and more effective chemotherapeutic agents.     

Development of the filaria Litomosoides galizai in the acarian vector

L. galizai develops in the gamasid B. bacoti in 11 days at 28 degrees C. Molts I and II take place respectively 5 and 7 days after feeding. The development is studied in the adult female mites; as for the laboratory filaria L. sigmodontis, the interstitial tissue is the main parasitized organ; microfilariae penetrate in the two cell types which constitute it: adipose cells and secretory cells (these secretory cells are described here for the first time). The filariae develop also in the salivary glands, the digestive wall, the genital envelopes and exceptionally in the coxal and vaginal glands. The filariae produce the formation of syncytia. Larvae which stay in the haemocele do not develop.     

Efficient generation of transgenic BALB/c mice using BALB/c embryonic stem cells

BALB/c is one of the most widely used and best characterized mouse strains in immunology. For various applications, it is necessary to generate BALB/c transgenic mice. However, using the conventional microinjection technique it is extremely inefficient to produce transgenic BALB/c mice since the one-cell stage BALB/c embryos are highly vulnerable to pronuclear DNA microinjection. To overcome this problem, we have investigated the generation of Egr-1 (early growth response gene) transgenic mice via the transfection of BALB/c embryonic stem cells. Transfectants carrying Egr-1 constructs comprising either the immunoglobulin heavy chain or the MHC class II promoter/enhancer system were injected into C57BL/6 host blastocysts resulting in chimeric mice. For both type of expression vectors, transgenic offspring of the germline chimeras expressed recombinant Egr-1 in lymphoid tissues containing B cells. This demonstrates the successful generation of Egr-1 transgenic BALB/c mice using transfected ES cell.     

Vaccine-induced immunity against cutaneous leishmaniasis in BALB/c mice.

Partially purified antigens, derived from Leishmania infantum or L. major promastigotes and isolated under reducing conditions, were used to immunize BALB/c mice. Three subcutaneous injections of the 64- to 97-kilodalton preparation in conjunction with muramyl dipeptide conferred long-lasting immunity against L. mexicana subsp. mexicana and L. major infection; they led to the development of antibodies neutralizing the infectiousness of promastigotes, induced specific delayed-hypersensitivity reactions, and generated populations of peritoneal macrophages capable of killing amastigotes. Vaccination resulted in no harmful effects, since these antigen neither exacerbated preexisting Leishmania infection nor impeded the formation of antibodies to other antigens administered concomitantly.     

Immune responses in BALB/c mice following immunization with aromatic compound or purine-dependent Salmonella typhimurium strains.

Two near isogenic strains of Salmonella typhimurium HWSH, stably mutated in either the aroA gene affecting the biosynthesis of aromatic compounds, or the purA gene affecting the biosynthesis of purines, were administered intravenously as live attenuated vaccines to BALB/c mice. HWSH aroA-immunized mice were well protected against intravenous (i.v.) challenge with wild-type virulent HWSH for at least 10 weeks, whereas HWSH purA-immunized mice were unprotected. Furthermore, HWSH aroA-immunized mice could also control a heterologous challenge with virulent Listeria monocytogenes at 7 and 14 days post-immunization, whereas mice receiving a similar dose of HWSH purA could not. Increasing the i.v. dose of HWSH purA compared to HWSH aroA induced some resistance to L. monocytogenes. Induction of early anti-S. typhimurium resistance by HWSH aroA immunization appeared slightly later than the anti-L. monocytogenes resistance. Mice immunized with either vaccine were able to mount S. typhimurium-specific T-cell proliferative responses and produced anti-S. typhimurium humoral antibodies in their serum. The antibody titre was greater in those mice immunized with the aroA mutant.     

Mycobacterium avium infection in BALB/c and SCID mice.

BALB/c and severe combined immunodeficient (SCID) mice were inoculated intraperitoneally with Mycobacterium avium and the numbers of cfu were monitored for 70 days in spleen, liver, lung, kidney, brain and peritoneum. While BALB/c mice formed typical granulomas and controlled bacterial growth in organs, a delay in development of lesions and a modest containment of infection were observed in SCID mice. In the spleen of BALB/c mice, in which bacterial growth was contained, macrophages (Mo) and natural killer (NK) cell numbers increased > or = 4.2 times and T- and B-cell numbers increased > or = 1.8 times after 42 days of infection; conversely, a low recruitment of mononuclear cells was observed in the spleen of SCID mice, where M. avium proliferated efficiently. Unlike visceral organs, a pronounced decrease in the number of cfu was observed in the peritoneum of BALB/c mice, concomitantly with a > or = 31.7-fold increase in Mo and NK cells and a > or = 9.1-fold increase in T and B cells. In the peritoneum of SCID mice only a bacteriostatic effect was observed despite a > or = 56.7-fold increase in Mo and NK cells and a > or = 22.3-fold increase in T and B cells. These results suggest that while an intact immune response can efficiently control M. avium infection in the spleen and peritoneum of BALB/c mice, cells of the innate immune system such as Mo and NK cells play a role in the containment of bacterial growth in the peritoneum, but not spleen, of SCID mice.     

Natural autoantibodies in nude and normal outbred (Swiss) and inbred (BALB/c) mice

Spleen cells from adult unprimed outbred (Swiss) and inbred (BALB/c) mice, either normal (no) or athymic-nude (nu) as well as spleen cells from Swiss nude mice bearing two different human tumors (BUR and PINQ), were fused with the mouse non-secreting myeloma cell line P3X63 Ag8-653. The supernatants of immunoglobulin secreting hybrids, all containing IgM, were screened for antibody activity against macromolecular antigens (autologous: actin, tubulin, myosin, dsDNA) and haptens (TNP, NP, NIP and NBrP). Furthermore, their idiotypic determinants were analyzed using a rabbit anti-idiotype which recognizes a major cross-reactive idiotype (IdD23) of BALB/c natural polyreactive autoantibodies. In all the mice studied, we identified: (1) hybrids reacting strongly with one or more haptens (10.7 to 37.8%) and (2) hybrids secreting natural monoclonal autoantibodies (NMoAb) with broad reactivities (polyreactive and/or oligoreactive) against autoantigens and/or haptens (11.4 to 26.8%). The results indicate that: (1) cells secreting natural autoantibodies with broad reactivities exist in both normal and nude mice, independently of the genetic background (inbred/outbred) of the mouse. However, in nude mice, the natural autoantibodies exhibit a more restricted pattern of reactivity (oligoreactive) compared to those of normal mice, and do not express the common idiotype IdD23 of natural polyreactive autoantibodies. (2) Tumors grafted into nude mice seem to induce the expression of polyreactive autoantibodies bearing the IdD23.     

HK-ATPase expression in the susceptible BALB/c and the resistant DBA/2 strains of mice to autoimmune gastritis

Neonatal thymectomy (NTx) in mice induces a group of alterations in the immune system homeostasis that results in the development of a variety of organ-specific autoimmune diseases such as gastritis, thyroiditis, oophoritis and orchitis. Given the importance of self-antigen expression in thymus for the control of autoreactive cells and generation of regulatory cells, we have compared the expression of parietal cell antigen in two strains of mice with the same H-2: BALB/c (susceptible to develop gastritis after NTx) and DBA/2 (resistant). We detected mRNA of HK-ATPase alpha and beta chains in day 1 thymi of both strains. Fifty percent of BALB/c mice presented mRNA levels similar to DBA/2. However, lower mRNA levels were found in the remaining BALB/c mice that may correspond to those that would develop AIG after NTx. Since the presence of the antigen in periphery is also necessary for the induction of regulatory cells, we have compared both strains observing in day 1 stomachs from resistant DBA/2 strain, a significantly higher content of positive cells for HK-ATPase subunits than stomachs from susceptible BALB/c strain. Also, the presence of antinuclear Abs in NTx BALB/c mice makes this model a useful experimental system for analyzing the responsible mechanisms breaking the non-specific self-tolerance.     

Nocardia infections in congenitally athymic (nude) mice and in other inbred mouse strains.

The mortality rate and histopathological features of Nocardia asteroides and Nocardia brasiliensis infections in congenitally athymic (nude) mice of ICR and C3H/eB origins were quite different from what we found for Swiss white mice and other inbred mouse strains (namely, C57/BL/6J, New Zealand Black, BALB/c, CBA/LAC, and C3H/eB). The immunocompetent littermates of the congenitally athymic mice occupied an intermediate position between their athymic siblings and Swiss white mice in terms of their responses to both these organisms. Macrophage ingestion and destruction of N. brasiliensis, as demonstrated by electron microscopy, was found to occur. The T-lymphocyte appears to be an essential component in normal mouse resistance to infection by both N. asteroides and N. brasiliensis.     

Experimental Porphyromonas gingivalis infection in nonimmune athymic BALB/c mice.

The purpose of this report was to study the role of T lymphocytes following injection of Porphyromonas gingivalis in a mouse abscess model. Three invasive P. gingivalis isolates (ATCC 53977, W83, and AJW4) were injected into athymic BALB/c mice and their heterozygous (nu/+) littermates. The athymic BALB/c (nu/nu) mice were able to localize the invasive P. gingivalis isolates at the injection site. By comparison, the heterozygous BALB/c (nu/+) littermates developed hemorrhagic secondary lesions within 24 h after subcutaneous injection of the same invasive P. gingivalis isolates. These results suggest that naive T lymphocytes may contribute to the pathology associated with P. gingivalis infection.