Nocardia asteroides and Nocardia brasiliensis infections in mice.

A model for Nocardia asteroides and Nocardia brasiliensis infections in Swiss white mice has been established without the addition to the inocula of any form of adjuvant. Serial histopathological studies revealed that these two actinomycetes cause lesions that are quite different in their features. An acute suppurative abscess characterizes the lesions of N. asteroides. In the case of N. brasiliensis infections a granuloma is produced in which a striking feature is the presence of large numbers of foam-laden macrophages, although occasional exceptions to this pattern were noted. Electron microscopic studies demonstrated that these macrophages contain within their cytoplasm organisms in varying stages of degeneration. Repeated mortality studies in mice failed to demonstrate differences in mortality rates produced by N. asteroides and N. brasiliensis. Thus, despite relatively trivial biochemical and antigenic differences between these two species of Nocardia, the local pathogenic response is quite different. The presence in the "brasiliensis lesion" of foamy macrophages with intracellular organisms is reminiscent of the histopathological features of lepromatous leprosy and of disseminated Myocobacterium bovis infection when this occurs in the immune suppressed situation. It is possible that N. brasiliensis infection produces a depression of cellular immunity that modifies the local host response to the organism.     

Adoptive transfer of immunity to Nocardia asteroides in nude mice.

Nude mice on a BALB/c background were adoptively transferred with unprimed spleen cells, Nocardia-primed spleen cells, or Nocardia-primed splenic T lymphocytes from syngeneic, heterozygous (nu/+) littermates. Two days later, these recipient mice and unmanipulated (control) nude mice were infected intravenously with a 50% lethal dose of Nocardia asteroides GUH-2 from an early stationary-phase culture. Antibody titers, spleen weights, percent mortality, and organ clearance of the microorganisms were measured at 3 h to 28 days after infection. Adoptively transferred nude mice had larger spleens and greater titers of anti-nocardial antibody 7 to 28 days after infection as compared with control nude mice. Adoptive transfer with either primed spleen cells or primed splenic T lymphocytes enhanced both the survival of recipient nude mice and their ability to eliminate N. asteroides from the liver and spleen. These data indicate that adoptive immunity to infection with N. asteroides can be transferred with either specifically primed spleen cells or splenic T lymphocytes. Thus, it appears that cell-mediated immunity and T lymphocytes are of uppermost importance in host resistance to nocardial infection.     

Monoclonal antibodies to Nocardia asteroides and Nocardia brasiliensis antigens.

Nocardia asteroides and Nocardia brasiliensis whole-cell extracts were used as antigens to generate monoclonal antibodies (MAbs). Six stable hybrid cell lines secreting anti-Nocardia spp. MAbs were obtained. These were characterized by enzyme-linked immunosorbent assay, Western blot (immunoblot), and immunofluorescence assay. Although all the MAbs exhibited different degrees of cross-reactivity with N. asteroides and N. brasiliensis antigens as well as with culture-filtrate antigens from Mycobacteria spp., they have the potential for use as reagents in the purification of Nocardia antigens.     

The use of immunodeficient male (CBA/N x BALB/c) F1 mice to produce monoclonal antibodies directed to proteins of Leptospira interrogans rather than to immunodominant lipopolysaccharides.

A method, using an immunodeficient mouse strain, for the production of monoclonal antibodies directed exclusively against the proteins in an antigen mixture also containing immunodominant LPS, is described. Male (CBA/N x BALB/c) F1 mice were immunized with an outer envelope antigen mixture from Leptospira interrogans strain Wijnberg containing both lipopolysaccharides and proteins. The immune response in these mice was shown to be predominantly directed against protein antigens. Hybridoma cell lines were generated by fusing spleen cells from a (CBA/N x BALB/c) F1 mouse with BALB/c Sp2/0 plasmacytoma cells. Hybridoma cell lines producing monoclonal antibodies reacting with the outer envelope preparation were identified by ELISA. All epitopes recognized by the monoclonal antibodies are sensitive to proteinase K degradation and resistant to oxidation by periodate indicating that they are located on proteins. All epitopes are located on a 35 kDa protein and specific for the pathogenic L. interrogans species.     

Effect of cyclophosphamide on experimental Nocardia asteroides infection in mice.

It was found that cells of Nocardia asteroides GUH-2 (virulent) were approximately 10 times more virulent than cells of N. asteroides 14759 (intermediate) and greater that 500 times more virulent than N. asteroides 10905 (avirulent) cells when early-stationary-phase cultures suspended in saline were injected intravenously into "normal" mice. There appeared to be a specific organ tropism for each strain. Thus, N. asteroides GUH-2 infected primarily the kidneys, N. asteroides 14759 infected the lungs and heart, and N. asteroides 10905 (in large doses) infected the lungs. Cyclophosphamide treatment of the mice 72 h prior to infection dramatically increased host susceptibility to nocardial infection, especially to N. asteroides 14759. However, cyclophosphamide treatment did not significantly alter the organ specificity for each strain. Cyclophosphamide greatly enhanced the ability of the nocardial strain to grow within its target organ and significantly altered normal host clearance from these organs.     

Complete Freund adjuvant treatment of pregnant females influences resorption rates in CBA/J x DBA/2 matings via progesterone-mediated immunomodulation.

Treatment of pregnant CBA/J females with CFA at day 0.5 and 7.5 of pregnancy significantly reduced the fetal resorption rates from 45% to 29% (P less than 0.05). Supernatants of progesterone-treated spleen cells from CFA treated CBA/J females pregnant of DBA/2 males significantly reduced natural cytotoxicity, while those of untreated identically pregnant mice had no effect. Supernatants of CFA-treated virgin mice blocked natural cytotoxicity to the same extent as those of CFA-treated pregnant mice. These data suggest that nonspecific immunostimulation induces progesterone receptors in spleen cells of CBA mice and that these receptors allow a progesterone dependent suppressive pathway to exert an antiresorptive effect.     

The "patchy" immunodeficiency of CBA/N mice.

The in vivo responses of CBA/N (which have an X-linked defect of B lymphocyte differentiation) and CBA/J normal mice to 2,4,6-trinitrophenylated (TNP) bacteriophage T4 and Diplococcus pneumoniae CS variant have been compared. Both antigens are thymus-independent (TI), and TNP-CS additionally contains the phosphorylcholine (PC) epitope, CBA/N and CBA/J mice give TNP plaque-forming cell responses similar in magnitude and avidity although only CBA/J give a PC response when challenged with TNP-CS. These results indicate that CBA/N mice have a "patchy" defect of antigen-induced humoral responses. This defect is manifested only in certain subpopulations of B cells and is not restricted to TI responses.     

FUNCTIONAL ANTIGEN BINDING BY THE DEFECTIVE B CELLS OF CBA/N x C3H/HeN F1 MALE MICE

CBA/N mice have an X-linked B cell defect which prevents them from responding to non-mitogenic thymic independent (TI-II) antigens such as dinitrophenylated (DNP-AGG) Ficoll. The F₁ male progeny of CBA/N female mice express the same defect. Spleen cell suspensions from such defective mice (CBA/N X C3H/HeN F₁ males) could not respond to DNP-AGG-Ficoll following in vitro immunization and subsequent transfer into irradiated, syngeneic, F₁ male recipients as expected. In contrast, normal CBA/N X C3H/HeN F₁ female spleen cells could respond and effect a ‘rescue'; they mounted strong plaque-foriming cell 7 days after in vitro exposure to DNP-AGG-Ficoll and subsequent transfer into irradiated F₁ male recipients. Defective F₁ male spleen cells could bind significant quantities of DNP-AGG-Ficoll, however, after, in vitro exposure. Extensive washing of these spleen cells could not reverse this binding. Such DNP-AGG-Ficoll-exposed and washed F₁ male spleen cells could, after transfer, aid normal untreated F₁ female cells in their rescue function. The defective F₁ male spleen cells could convey immunogenic quantities of DNP-AGG-Ficoll to the ‘rescuing’ F₁ female cells. Mitomycin treatment of F₁ male cells did not interfere with their conveyor function. Goat anti-mouse μ serum impeded the passive antigen conveyor function of defective F₁ male cells as did prior exposure to high concentrations of free DNP-AGG hapten. Our data support the view that the B cell defect of CBA/N X C3H/HeN F₁ male mice does not relate to antigen binding, but rather to an inability to be effectively triggered by certain cell-bound polymeric antigens.     

Frequencies of mitogen-reactive B cells in the mouse. Lipopolysaccharide-, lipoprotein- and Nocardia mitogen-reactive B cells in CBA/N mice.

B cell-defective (CBA/N X BALB/c)F1 male mice have 100 times less lipopolysaccharide-reactive and 10--100 times less lipoprotein- and Nocardia mitogen-reactive B cell precursors than F1 female mice with normal B cells.     

Immunological responses to protein, carbohydrate and lipid fractions of Nocardia asteroides in mice

The protein, polysaccharide and phospholipid constituents of Nocardia asteroides have been partially purified and their immunogenicity studied in mice. Humoral and cellular immune responses were demonstrated against a crude cytoplasmic protein fraction (CPF). Two fractions of CPF were prepared on a Sephadex G-200 column; a high mol. wt fraction, fraction-1(F1) was capable of eliciting both types of immune responses, whereas fraction-2(F2) behaved more like a hapten. Phosphatides elicited only humoral responses whereas polysaccharides were non-immunogenic.     

Therapy with dendritic cells influences the spontaneous resorption rate in the CBA/J x DBA/2J mouse model

PROBLEM: DBA/2J-mated CBA/J female mice are prone to a high incidence of fetal abortions. This fetal wastage can be dramatically reduced by immunizing the female mice with BALB/c, but not with DBA/2J spleen cells during early gestation. Nevertheless, the underlying mechanisms remain to be elucidated. Recently, dendritic cells (DC) have been described at the feto-maternal interface in the human uterus. In this work, we studied the effect of adoptive transfer of DC on the maintenance of pregnancy in the CBA/J x DBA/2J model. METHODS: Bone marrow-derived DC were generated from virgin female CBA/J mice (6-8 weeks old). CBA/J females were inoculated with DC twice before mating. Four different experimental groups were included: (i) no treatment control, (ii) mice injected with culture medium [granulocyte-macrophage colony-stimulating factor (GM-CSF)], (iii) immunized with DC and (iv) immunized with paternal DBA/2J antigens lisate-pulsed DC, n = 5. RESULTS: The control abortion rate was 23.8%, and with GM-CSF alone was 17.6%. Following inoculation of syngeneic DC abortion rates were reduced to 2.2%, but protection was short-lived. Abortion rates with DC pulsed with DBA/2J antigens was 5%. Serum of interleukin (IL)-6 levels were lower in the latter two groups up to the time of abortion. The kinetics of immunoglobulin G asymmetric antibodies synthesis was modified, but there was no correlation between asymmetric antibodies production and the lowering of abortions rates. CONCLUSION: Syngeneic DC prevented abortions and this was linked to a decrease in IL-6 levels, but not with levels of asymmetric antibodies.     

L-dopa-responsive movement disorder caused by Nocardia asteroides localized in the brains of mice.

Nocardia asteroides can cause infections in the brain of humans and a variety of animals. In mice, invasion of the central nervous system results in specific neurologic signs. Following intravenous injection of various doses of log-phase N. asteroides GUH-2 into female BALB/c mice, localization and growth of nocardial cells within the brains were determined, histopathological sections were prepared, and Nissl substance and tyrosine hydroxylase immunoreactivity were observed. Mice were monitored for the development of neurologic signs, and their responsiveness to L-dopa was determined. It was shown that nocardial cells became localized within specific regions of the brain and then underwent rapid growth followed by a delayed clearance, and there was no inflammatory response at the site of invasion for 24 h. Mice that received a subclinical dose of nocardiae developed specific neurologic signs that emerged following the elimination of nocardial cells from the brain. On the basis of the specific signs, mice could be divided into distinct groups. One group consisted of animals that had a form of hemiparesis that did not respond to L-dopa. They expressed a deviation of the head and a tendency to roll, and when suspended by the tail they would spin rapidly. The second group of mice developed a rhythmic, uncontrolled vertical shake of the head (four to five times per s) tremulous movement, stooped posture, restlessness, and no signs of hemiparesis. The head shakes were temporarily stopped by treatment with L-dopa. Mice that expressed head shakes had a loss of Nissl substance and tyrosine hydroxylase immunoreactivity in the neurons of the substantia nigra and ventral tegmental areas of the brain. Hyaline inclusion bodies that resembled Lewy bodies were found in the neurons of mice with head shake 1 month after infection. Therefore, mice infected with N. asteroides may serve as a model for studying parkinsonian signs and other degenerative diseases involving extrapyramidal and pyramidal systems.     

Demonstration of T15 idiotype-positive effector and suppressor T cells for phosphorylcholine-specific delayed-type hypersensitivity response in CBA/N or (CBA/N X BALB/c)F1 male mice

Subcutaneous immunization of CBA/N or (CBA/N x BALB/c)F₁ (NBF₁) male mice, which were defective in phosphorylcholine (PC)-specific humoral antibody response, with PC-conjugated syngeneic spleen cells induced a PC-specific delayed-type hypersensitivity (DTH) response, while intravenous administration of the same cells induced PC-specific suppressor T cells for the DTH response. Treatment of PC-specific effector or suppressor T cells for the DTH response induced in NBF₁ male mice with anti-T15 idiotypic antibody inactivated effector or suppressor functions of these T cells implying that PC-specific T cells in NBF₁ mice expressed T15 idiotypic determinants on their surface. Enrichment of PC-specific suppressor T cells was also shown by employing anti-T15 antibody-coated dishes.     

Virulence of Nocardia asteroides during its growth cycle.

Cells of Nocardia asteroides undergo structural and chemical changes, especially in the cell wall, during growth in brain heart infusion broth. Experiments were devised to determine whether these changes affected the virulence of Nocardia for mice. It took, on the average, 1,380 times the number of colony-forming units at the stationary phase to achieve the same mortality induced by the log-phase cells. Cells in either the lag phase or early stationary phase of growth were intermediate in the numbers of colony forming units required to kill mice. Dry-weight determinations at different stages of growth demonstrated that the log-phase organisms were approximately 10 times heavier than stationary-phase cells. Thus, on the basis of dry-weight (micrograms) values, the average colony-forming unit of log phase is approximately 130 times more virulent than in stationary-phase cultures. Therefore, the stage of growth affects greatly the virulence of N. asteroides.     

DNA probes for the identification of Nocardia asteroides.

DNA probes for the rapid identification of Nocardia asteroides were obtained by constructing a genomic library of strain GUH-2 in the lambda cloning vector EMBL3. Of 50 recombinant clones tested, 2 were identified that hybridized with 31% of the N. asteroides strains in a reference collection without cross-hybridization with related members of the Actinomycetales. Additional libraries were then generated from selected strains of N. asteroides that had failed to hybridize with any of the GUH-2 clones. Four additional clones were obtained from these strains which, when pooled, provided DNA probes specific for all of the N. asteroides strains tested.     

Adherence of Nocardia asteroides within the murine brain.

Nonlethal infection of BALB/c mice with Nocardia asteroides GUH-2 (GUH-2) produces a variety of neurological signs, including an L-dopa-responsive movement disorder in 10 to 15% of the infected population. To study nocardial interactions with the brain, we characterized the attachment of GUH-2 within specific regions through the use of microdissection. Following an intravenous injection of a single-cell suspension of log-phase GUH-2, viable cells were recovered from all regions of the brain, and the distribution of the nocardiae was independent of the size of the inoculum. In addition, two mutants of GUH-2 were found to possess significantly altered binding characteristics with regard to both the percentage of the inoculum bound per brain and the relative distribution of adherence to regions of the brain, when compared with the parental strain. These results indicated that GUH-2 bound throughout the murine brain and suggested that GUH-2 utilized specific receptors to facilitate this attachment.     

Relationship of macrophages to cell-mediated immunity in experimental Nocardia asteroides infection.

Marked in vivo intracellular killing of Nocardia asteroides occurred in the peritoneal macrophages obtained 72 h after an intraperitoneal challenge with N. asteroides, in guinea pigs either actively immunized with ribonucleic acid protein or passively immunized by immune spleen cell transfer from actively immunized donor guinea pigs. This specific killing of N. asteroides in immune macrophages persisted for at least up to 60 days. Administration of antimacrophage sera before intravenous challenge with N. asteroides in the immune guinea pigs produced an early death of the animals, and the total tissue counts of N. asteroides in the liver, spleen, lungs, and heart remained the same in them as in unimmunized controls.     

Spontaneous myocarditis in DBA/2 mice

DBA/2 inbred mice spontaneously develop myocarditis and a unique form of subepicardial inflammation of the right ventricle characterized by a prominent eosinophilic infiltrate with calcinosis. We studied this myocarditis using light microscopy and both transmission and analytical X-ray electron microscopy, paying particular attention to eosinophil-associated cardiocyte injury. At 5 weeks of age, many eosinophils and mononuclear cells (MNCs) were seen in the subepicardium of the right ventricle. Electron microscopy showed that cardiocytes underwent degenerative changes, including myofibrillar lysis, accumulation of Z-band material and mitochondrial inclusions, and rupture of plasma membranes. The infiltrating eosinophils appeared to be activated, and cells with cytoplasmic vacuoles, suggestive of degranulation, were noted. The myocardial injury was most severe in the 7th week and healed with myocardial fibrosis and calcinosis by the 8th week. Analytical X-ray electron microscopy showed that the calcinosis was initiated in mitochondrial inclusions of injured cardiocytes. The peripheral eosinophil count did not increase during the course of the disease, but there was a positive correlation between the ratio of eosinophils to infiltrated white blood cells (Eo/WBCs) in the right ventricle and the severity of myocardial damage. Eosinophils may play a significant part in subepicardial cardiocyte injury seen in DBA/2 mice.