Higher frequency of Leishmania major-specific L3T4+ T cells in susceptible BALB/c as compared with resistant CBA mice

In previous studies, we reported that a) the adoptive transfer of parasite-specific L3T4+ T cells enhanced rather than inhibited the development of lesions induced by Leishmania major in normal BALB/c mice, and b) the depletion in vivo of L3T4+ T cells by administration of anti-L3T4 monoclonal antibody reversed the susceptibility of BALB/c mice to L. major. To further assess the role of specific L3T4+ T cells in the development of lesions induced by L. major in BALB/c mice, the frequency of parasite-specific T cells capable of mediating specific delayed-type hypersensitivity (DTH) reactivity was determined, by limiting dilution analysis, in the lymph nodes draining the lesions of susceptible (BALB/c) and resistant (CBA) mice. The numbers of L. major-specific DTH-mediating T cells was found to be substantially increased in the lymph nodes of infected BALB/c mice as compared with CBA mice. Moreover in CBA mice, analysis of the cell surface phenotype of the L. major-specific DTH-mediating T cells showed that these cells were equally represented in the L3T4+, Lyt-2-, and L3T4- Lyt-2+ subsets, whereas the majority of these cells in BALB/c mice expressed the L3T4+ Lyt-2- surface phenotype.     

Isolation of protective T cells from BALB/cJ mice chronically infected with Leishmania donovani

BALB/cJ mice, which are homozygous for Lshs on chromosone 1, are genetically susceptible to Leishmania donovani (S3), but spontaneously reduce their parasite burdens late in the course of infection. Spleens from chronically infected mice (5 to 8 mo) were found to have T cells that responded to leishmanial Ag by proliferating and secreting immune IFN. An IFN-secreting T cell line derived from the spleen of a chronically infected mouse, after boosting with leishmanial Ag and treatment with Pentostam to kill residual parasites, was able to activate macrophages to kill amastigotes in vitro. Furthermore, after adoptive transfer of this cell line, naive BALB/cJ mice challenged with amastigotes demonstrated a 42-fold reduction in parasite burden compared to controls. Four of five clones derived from the protective T cell line secreted IFN and proliferated between days 7 and 14 after Ag stimulation. All clones were Ly1+2-, L3T4+. Another T cell line, which had Ly1+2-, L3T4+ phenotype, was derived from the lymph nodes of s.c. immunized, uninfected mice. It multipled but never produced IFN in response to leishmanial Ag and failed to protect macrophages against L. donovani infection in vitro or in vivo. This nonprotective T cell line and the fifth clone from the protective line, which also did not secrete IFN, proliferated between days 2 and 7 after Ag stimulation. In summary, leishmania-responsive helper/inducer T cells, which produced IFN but were slow to proliferate in response to Ag in vitro and which were able to activate macrophages to reduce amastigotes in vitro and in vivo, are present in chronically infected BALB/cJ mice and may mediate the decrease in parasite burden during chronic infection.     

BALB/c mice produce blister-causing antibodies upon immunization with a recombinant human desmoglein 3

Pemphigus vulgaris (PV) is an Ab-mediated autoimmune blistering disease of mucotaneous surfaces. Over 95% of the patients with PV express DR4 or DRw6, and the disease is characterized by the presence of autoantibodies directed against desmoglein 3 (Dsg 3), a protein expressed on keratinocytes. An appropriate animal model is required to understand immunoregulation and to address the role of immunogenetic components in the production of pathogenic Abs that are characteristic of PV. Therefore, we turned to the development of a mouse model. Four strains of female mice (BALB/c, DBA/1, SJL/J, and HRS/J) were screened for their ability to produce pathogenic anti-Dsg 3 Abs. We demonstrated that only BALB/c mice immunized with a full-length Dsg 3 can produce pathogenic Abs capable of causing acantholysis of human foreskin in culture and blistering in neonatal mice. This observation suggested that either H-2d or the BALB background contains the immunogenetic makeup necessary for the production of pathogenic anti-Dsg 3 Abs. No correlation was noted between a given isotype and the pathogenic potential of autoantibodies from different strains of mice. Similarly, the pattern of reactivity of Abs with a panel of 46 synthetic peptides that span the entire Dsg 3 failed to reveal any association between binding specificity and the pathogenic potential, and suggested that pathogenic Abs might recognize conformational epitopes. Moreover, our studies showed that the epitopes recognized by pathogenic Abs are contained within the extracellular Dsg 3.     

Differentiated cells from BALB/c mice differ in their radiosensitivity

Various isolated cells of an inbred mouse strain (BALB/c) differed widely in their sensitivity to gamma irradiation: fibroblasts are five times more resistant than peripheral lymphocytes. Among lymphocytes, T cells are more resistant than B cells. Cell lines derived from the primary cells conserved their radiosensitivity. Cytofluorometric measurements show that the differential reaction of a cell to gamma irradiation can be detected already 2–3 h after the irradiation event. Radiation-sensitive cells are delayed for a longer time in S phase and G2 phase of the cell cycle than radiation-resistant cells. No difference in the capacity of the cells to perform single-strand break repair, double-strand break repair or unscheduled DNA synthesis could yet be detected.     

Altered kinase C function in transformed BALB/c3T3 cells

Comparative studies of kinase C function were performed in an untransformed (A31) and the benzo[a]pyrene (BPA31), dimethylbenz[a]anthracene (DA31), and Kirsten sarcoma virus (KA31) transformed BALB/c 3T3 mouse fibroblast cell lines. The 80-kDa kinase C dependent phosphoprotein (pp80), an in vivo marker of kinase C activity, was markedly decreased in the transformed cells although the amount of the 80-kDa substrate protein in the BPA31 cells was similar to that in the untransformed A31 cells. Total cell lysate kinase C levels were lower in the transformed cells but this difference could not account for the reduced pp80 phosphorylation. Increased affinity of kinase C for the membrane fraction in the BPA31 cells may account for decreased phosphorylation of pp80.     

Effects of a bacteriocin from Mycobacterium smegmatis on BALB/3T3 and simian virus 40-transformed BALB/c mouse cells

The effects of a bacteriocin from Mycobacterium smegmatis ATCC 14468 on simian virus 40-transformed BALB/c mouse cells (mKS-A TU-7 cells) and nontransformed BALB/3T3 cells originating from the BALB/c mouse strain were studied. The percentage of nigrosin-unstained (viable) cells in the bacteriocin-treated mKS-A TU-7 cells decreased time-dependently with an increase in the bacteriocin activity. There was a time-dependent decrease in the bacteriocin activity after treatment with the cell membrane preparation from mKS-A TU-7 cells. There was no apparent effect of the bacteriocin on the viability of nontransformed BALB/3T3 cells. Wheat germ agglutinin blocked the toxic effect of bacteriocin on mKS-A TU-7 cells. These results indicate that the higher sensitivity and binding capacity of the tumor cells to the bacteriocin is probably due to the presence of a large amount of N-acetyl-glucosamine or closely related sugar residues with a high affinity for bacteriocin, as compared with normal cells. The bacteriocin produced morphological alterations and inhibition of synthesis of ribonucleic acid, deoxyribonucleic acid and protein in the transformed but not in the nontransformed cells.     

Deletion of IL-4Ralpha on CD4 T cells renders BALB/c mice resistant to Leishmania major infection

Effector responses induced by polarized CD4+ T helper 2 (Th2) cells drive nonhealing responses in BALB/c mice infected with Leishmania major. Th2 cytokines IL-4 and IL-13 are known susceptibility factors for L. major infection in BALB/c mice and induce their biological functions through a common receptor, the IL-4 receptor alpha chain (IL-4Ralpha). IL-4Ralpha-deficient BALB/c mice, however, remain susceptible to L. major infection, indicating that IL-4/IL-13 may induce protective responses. Therefore, the roles of polarized Th2 CD4+ T cells and IL-4/IL-13 responsiveness of non-CD4+ T cells in inducing non-healer or healer responses have yet to be elucidated. CD4+ T cell-specific IL-4Ralpha (Lck(cre)IL-4Ralpha(-/lox)) deficient BALB/c mice were generated and characterized to elucidate the importance of IL-4Ralpha signaling during cutaneous leishmaniasis in the absence of IL-4-responsive CD4+ T cells. Efficient deletion was confirmed by loss of IL-4Ralpha expression on CD4+ T cells and impaired IL-4-induced CD4+ T cell proliferation and Th2 differentiation. CD8+, gammadelta+, and NK-T cells expressed residual IL-4Ralpha, and representative non-T cell populations maintained IL-4/IL-13 responsiveness. In contrast to IL-4Ralpha(-/lox) BALB/c mice, which developed ulcerating lesions following infection with L. major, Lck(cre)IL-4Ralpha(-/lox) mice were resistant and showed protection to rechallenge, similar to healer C57BL/6 mice. Resistance to L. major in Lck(cre)IL-4Ralpha(-/lox) mice correlated with reduced numbers of IL-10-secreting cells and early IL-12p35 mRNA induction, leading to increased delayed type hypersensitivity responses, interferon-gamma production, and elevated ratios of inducible nitric oxide synthase mRNA/parasite, similar to C57BL/6 mice. These data demonstrate that abrogation of IL-4 signaling in CD4+ T cells is required to transform non-healer BALB/c mice to a healer phenotype. Furthermore, a beneficial role for IL-4Ralpha signaling in L. major infection is revealed in which IL-4/IL-13-responsive non-CD4+ T cells induce protective responses.     

Effects of Trichinella spiralis infections upon bone marrow stem cells in BALB/c mice

Trichinella spiralis infections provoke a variety of responses in the host, some of which involve stem cell proliferation and myeloid cell maturation, increases in the mast cell precursor cell populations, and maturation and eosinopoiesis. Very little is known about the influence of T. spiralis upon bone marrow stem cells and splenic colony formation. In the present communication we report that T. spiralis infection in mice stimulates the generation of colony-forming units in the spleen (CFU-S). Passive transfer of bone marrow cells from uninfected BALB/c mice to X-irradiated (650 R) T. spiralis-infected recipients resulted in a significant increase of CFU-S at 14 and 24 days postinfection. Passive transfer of bone marrow cells from T. spiralis-infected mice to X-irradiated uninfected mice also resulted in increased numbers of CFU-S in the donor mice at 24 days postinfection. These findings strongly suggest that T. spiralis infection conditions the microenvironment in the spleen which stimulates CFU-S.     

Protective immunity against Leishmania major induced by Leishmania tropica infection of BALB/c mice

Leishmania (L.) tropica is a causative agent of human cutaneous and viscerotropic leishmaniasis. Immune response to L. tropica in humans and experimental animals are not well understood. We previously established that L. tropica infection induces partial protective immunity against subsequent challenge infection with Leishmania major in BALB/c mice. Aim of the present study was to study immunologic mechanisms of protective immunity induced by L. tropica infection, as a live parasite vaccine, in BALB/c mouse model. Mice were infected by L. tropica, and after establishment of the infection, they were challenged by L. major. Our findings shows that L. tropica infection resulted in protection against L. major challenge in BALB/c mice and this protective immunity is associated with: (1) a DTH response, (2) higher IFN-γ and lower IL-10 response at one week post-challenge, (3) lower percentage of CD4⁺ lymphocyte at one month post-challenge, and (4) the source of IFN-γ and IL-10 were mainly CD4⁻ lymphocyte up to one month post-challenge suggesting that CD4⁻ lymphocytes may be responsible for protection induced by L. tropica infection in the studied intervals.     

The opioid antagonist naloxone inhibits Leishmania major infection in BALB/c mice

BALB/c mice are susceptible to develop non-healing, progressive infection with Leishmania major (L. major) due to the development of a non-protective Th2 response. Resistance to L. major infection is dependent to Th1 response. Treatment of mice with the opioid antagonist naloxone can promote the activation of Th1 responses. Here we study the effect of chronic administration of various doses of naloxone on susceptibility of BALB/c mice to L. major infection. Our results showed that naloxone has dose-dependent biphasic effect on L. major infection in BALB/c mice. While administration of 1mg/kg × 2/day tends to exacerbate the local reaction to L. major infection, treatment with 10mg/kg × 2/day of naloxone suppresses the local reaction and progress of infection. On the other hand treatment of mice with middle dose (5mg/kg whether 1 or 2 times per day) does not have significant effect on the infection. This study demonstrates that administration of high dose of naloxone could improve protection against L. major infection in BALB/c mice, presumably by modulation in Th1/Th2 balance or by affecting macrophages through binding to Toll-like receptors.     

Signal-mediated nuclear transport in proliferating and growth-arrested BALB/c 3T3 cells

Mediated transport across the nuclear envelope was investigated in proliferating and growth-arrested (confluent or serum starved) BALB/c 3T3 cells by analyzing the nuclear uptake of nucleoplasmin-coated colloidal gold after injection into the cytoplasm. Compared with proliferating cells the nuclear uptake of large gold particles (110-270 A in diameter, including the protein coat) decreased 5.5-, 33-, and 78- fold, respectively, in 10-, 14-17-, and 21-d-old confluent cultures; however, the relative uptake of small particles (total diameter 50-80 A) did not decrease with increasing age of the cells. This finding suggests that essentially all pores remain functional in confluent populations, but that most pores lose their capacity to transport large particles. By injecting intermediate-sized gold particles, the functional diameters of the transport channels in the downgraded pores were estimated to be approximately to 130 and 110 A, in 14-17- and 21-d- old cultures, respectively. In proliferating cells, the transport channels have a functional diameter of approximately 230 A. The mean diameters of the pores (membrane-to-membrane distance) in proliferating and confluent cells (728 and 712 A, respectively) were significantly different at the 10%, but not the 5%, level. No differences in pore density (pore per unit length of membrane) were detected. Serum- deprived cells (7-8 d in 1% serum or 4 d in 0.5% serum) also showed a significant decrease in the nuclear uptake of large, but not small, gold particles. Thus, the permeability effects are not simply a function of high cell density but appear to be growth related. The possible functional significance of these findings is discussed.     

Lymph node cell responsiveness in BALB/c mice infected with Leishmania mexicana

In the present study we measured the blastogenic response of lymph node cells from BALB/c mice infected with Leishmania mexicana throughout the course of infection. Results showed that infected mice displayed normal blastogenic responses in the lymph nodes until twenty weeks of infection. Thereafter, there was a gradual suppression. Comparison of the immunoresponsiveness in the spleen and lymph nodes, revealed normal responses in the lymph nodes several weeks after suppression in the spleen had occurred. Suppression of blastogenic responses in the lymph nodes was related to an adherent macrophage-like cell which actively suppressed normal proliferative responses to mitogens.     

Protective effect of lectin from Synadenium carinatum on Leishmania amazonensis infection in BALB/c mice

The protective effect of the Synadenium carinatum latex lectin (ScLL), and the possibility of using it as an adjuvant in murine model of vaccination against American cutaneous leishmaniasis, were evaluated. BALB/c mice were immunized with the lectin ScLL (10, 50, 100 microgram/animal) separately or in association with the soluble Leishmania amazonensis antigen (SLA). After a challenge infection with 10(6) promastigotes, the injury progression was monitored weekly by measuring the footpad swelling for 10 weeks. ScLL appeared to be capable of conferring partial protection to the animals, being most evident when ScLL was used in concentrations of 50 and 100 microgram/animal. Also the parasite load in the interior of macrophages showed significant reduction (61.7%) when compared to the control group. With regard to the cellular response, ScLL 50 and 100 microgram/animal stimulated the delayed-type hypersensitivity (DTH) reaction significantly (P < 0.05) higher than SLA or SLA plus ScLL 10 weeks after the challenge infection. The detection of high levels of IgG2a and the expression of mRNA cytokines, such as IFN-gamma, IL-12, and TNF-alpha (Th1 profiles), corroborated the protective role of this lectin against cutaneous leishmaniasis. This is the first report of the ScLL effect on leishmaniasis and shows a promising role for ScLL to be explored in other experimental models for treatment of leishmaniasis.     

猪圆环病毒3型感染BALB/c小鼠的实验观察

目的研究猪圆环病毒3型(porcine circovirus3,PCV3)对BALB/c小鼠的感染情况。方法将雌性BALB/c小鼠分为两组,实验组感染PCV3组织毒,对照组接种同样剂量的PBS。感染后每天观察小鼠状态,并在第0,3,7,11和14天采血进行荧光定量PCR检测和ELISA检测。实验结束后,对所有动物进行安乐死和剖检,对心脏、肝、脾、肺、肾、脑和淋巴结取样进行荧光定量PCR检测,并制片进行组织病理学检查。选择PCR检测阳性组织进行PCV3CAP基因测序分析。结果PCV3组织毒感染小鼠不引起明显的临床症状和病理变化。病毒可在感染早期的血清中检测到,病毒含量最高的器官是肝脏和脾脏,PCV3感染小鼠后核酸序列未发生变化。随着感染时间的增加血清中抗体水平逐渐升高。结论PCV3可以感染BALB/c小鼠,并在小鼠体内增殖。本研究结果为猪圆环病毒3型致病性的研究以及防控提供了参考。     

Immune response to Leishmania (Leishmania) chagasi infection is reduced in malnourished BALB/c mice

Protein-energy malnutrition and micronutrient deficiencies may down-regulate immune response and increase morbidity and mortality due to infection. In this study, a murine model was used to study the effects of protein, iron and zinc deficiencies on the immune response to Leishmania (Leishmania) chagasi infection. Mice were initially fed a standard diet or with a diet containing 3% casein but deficient in zinc and iron. After malnutrition was established, mice were inoculated with L. chagasi and sacrificed four weeks later in order to evaluate liver and spleen parasite loads and serum biochemical parameters. Significant decreases in liver and spleen weight, an increase in the parasite loads in these organs and decreases in serum protein and glucose concentrations in malnourished animals were observed. Furthermore, the production of interferon-gamma by spleen cells from infected malnourished mice stimulated by Leishmania antigen was significantly lower compared with that in control diet mice. These data suggest that malnutrition alters the immune response to L. chagasi infection in the BALB/c model and, in association with the effects on biochemical and anatomical parameters of the host, favored increases in the parasite loads in the spleens and livers of these animals.     

The chemokine receptor CXCR3 attenuates the control of chronic Mycobacterium tuberculosis infection in BALB/c mice

The chemokine receptor CXCR3 plays a significant role in regulating the migration of Th1 cells. Given the importance of Th1 immunity in the control of tuberculous infection, the results of the present study demonstrating that CXCR3-deficient BALB/c mice are more resistant to Mycobacterium tuberculosis, compared with wild-type mice, is surprising. This enhanced resistance manifests in the chronic but not the acute phase of infection. Remarkable differences in the cellular composition of the pulmonic granuloma of the CXCR3(-/-) and wild-type mice were found, the most striking being the increase in the number of CD4(+) T cells in the knockout strain. In the chronic phase of infection, the number of CD69-expressing CD4(+) T lymphocytes in the lungs of CXCR3(-/-) mice was higher than in wild-type mice. Additionally, at 1 mo postinfection, the number of IFN-gamma-producing CD4(+) T cells in the lungs and mediastinal lymph nodes of the CXCR3-deficient strain was elevated compared with wild-type mice. Pulmonic expression of IFN-gamma, IL-12, TNF-alpha, or NO synthase 2, the principal antimycobacterial factors, were equivalent in the two mouse strains. These results indicate that: 1) CXCR3 plays a role in modulating the cellular composition of tuberculous granuloma; 2) CXCR3 impairs antimycobacterial activity in chronic tuberculosis; and 3) in the absence of CXCR3, mice exhibit a heightened state of CD4(+) T lymphocyte activation in the chronic phase of infection that is associated with enhanced CD4(+) T cell priming. Therefore, CXCR3 can attenuate the host immune response to M. tuberculosis by adversely affecting T cell priming.     

Overexpression of vinculin suppresses cell motility in BALB/c 3T3 cells.

The content of vinculin, a cytoplasmic protein found in focal contacts and cell-cell junctions, was increased in BALB/c 3T3 cells by gene transfection. The vinculin expressed from the full length chicken cDNA, incorporated into focal contacts and its pattern was identical to that of the endogenous protein. Cells stably expressing vinculin by 20% over the endogenous level had altered locomotory properties. In these cells, the ability to migrate into a wound formed in a confluent monolayer and the locomotion of individual cells were drastically reduced. The results provide direct evidence that cell locomotion can be regulated by modulating vinculin expression.     

Cross-reactivity of IgM-secreting B cells from normal BALB/c mice.

Cross-reactive antibodies capable of binding to foreign and self Ag are present in the serum of normal newborn and adult animals. In our work, a chamber ELISA assay was used to quantitate the cross-reactivity of B cells actively secreting Ig in BALB/c mice of different ages. Individual lymphocytes were tested for the production of IgM antibodies capable of binding to a series of four unrelated Ag (DNA, TNP, actin, and OVA). Results indicate that nearly one-quarter of IgM secreting lymphocytes from 6-day-old animals were cross-reactive. This frequency was two- to fourfold higher than that found in adult mice. Very old animals, however, showed a selective increase in the cross-reactivity of anti-DNA (but not anti-TNP) secreting lymphocytes. Evidence from Ag inhibition experiments indicated that low concentrations of soluble Ag could block the binding of polyreactive antibodies, and that approximately one-half of "naturally" cross-reactive B cells produced antibodies capable of binding to three or more unrelated Ag.     

Mitogenic and cytotoxic actions of tumor necrosis factor in BALB/c 3T3 cells. Role of phospholipase activation

In addition to its cytotoxic/cytostatic action on many tumor cells in vitro, tumor necrosis factor (TNF) was recently shown to stimulate the growth of some types of cells in culture. We examined the action of TNF in BALB/c 3T3 cells which have been used extensively to study growth regulation. In subconfluent, rapidly dividing 3T3 cultures, murine (Mu) TNF was cytotoxic, while human (Hu) TNF had virtually no antiproliferative action on the cells. In contrast, in density-arrested BALB/c 3T3 cells maintained in a chemically defined, serum-free medium, MuTNF produced a dose-dependent stimulation of DNA synthesis. In stimulating DNA synthesis, MuTNF acted synergistically with both epidermal growth factor or platelet-derived growth factor. While stimulating DNA synthesis in quiescent 3T3 cultures, high doses of MuTNF (100 or 10 ng/ml) were also cytotoxic for a portion of the cells in the same cultures. Cytotoxicity was apparent 2 h after the addition of MuTNF, well before the onset of DNA synthesis. BALB/c 3T3 cell variants selected for their resistance to the cytotoxic action of MuTNF retained the capacity to respond to the mitogenic action of MuTNF, indicating that the stimulation of DNA synthesis by TNF is not a consequence of a TNF "wounding effect." Addition of TNF to density-arrested 3T3 cells resulted in the release of free arachidonic acid and palmitic acid from the cells. Quinacrine, a phospholipase inhibitor, inhibited both cytotoxicity and DNA synthesis in response to TNF, and melittin, a phospholipase activator, mimicked both the cytotoxic and mitogenic actions of TNF in quiescent BALB/c 3T3 cells. These results suggest that phospholipid breakdown is part of the essential early signal transduction events required both for the cytotoxic and mitogenic response to TNF action.