Inhibition of murine humoral immune responses by substances derived from trypanosomes

Previous studies of depressed immune responses in mice infected with the mouse-specific Trypanosoma musculi have produced no evidence of major involvement of typical suppressor lymphocytes or macrophages. We continue this line of investigation in the present report by demonstrating that: a) T. musculi strongly suppress the responses of nude mouse spleen cells to the T-independent antigen, TNP-LPS; b) spleen cell preparations of infected mice display a substantial proportion of cells bearing trypanosome-derived substances (TDS) demonstrable by specific rabbit antibody against T. musculi (RATS); c) treatment of spleen cells from infected mice with RATS plus C eliminates the inhibitory effect of these spleen cells on the immune responses of co-cultivated normal spleen cells; d) incubation in vitro of normal spleen cells with an extract of T. musculi results in progressive loss of the cells to respond to antigens and, in addition, confers on the treated cells to respond to antigens and, in addition, confers on the treated cells the property of inhibiting the responses of co-cultivated normal spleen cells; e) T. lewisi, the rat-specific trypanosome, fails to inhibit murine immune responses. We conclude that the immunoinhibitory effects of T. musculi on murine immune responses are associated with the cytophilic binding of TDS (possibly in the form of immune complexes) and that this vigorous mechanism of inhibition will be shown to involve nonspecific mitogenic and/or biosynthetic activation of lymphocytes.     

The effect of cyclosporin A on Trypanosoma musculi infection of mice

Cyclosporin A treatment of mice infected with Trypanosoma musculi mimicked the effect of T-cell deprivation in increasing the parasitemia and lowering the antibody responses. However, in contrast to T-cell deprivation, cyclosporin A treated mice were still able to clear the parasites from the blood. As expected, T-cell deprived mice were not responsive to cyclosporin A. It is suggested that antibodies against T-independent antigens of the parasite are ineffective in promoting elimination of the trypanosomes. These findings support the importance of T-helper cells in the final elimination of T. musculi from the blood of mice.     

Efficacy of selamectin against mites (Myobia musculi, Mycoptes musculinus and Radfordia ensifera) and nematodes (Aspiculuris tetraptera and Syphacia obvelata) in mice

The effects of selamectin were studied in mice naturally infected with the mites Myobia musculi, Myoceptes musculinus and Radfordia ensifera and with the oxyurid nematodes Aspiculuris tetraptera and Syphacia obvelata. The mice were divided into three treated and three control groups (n=9). Selamectin in the range 10-12.4 mg/kg was applied topically to the skin in a single spot at the base of the neck in front of the scapulae. The mice of treated and control groups were necropsied on the 4th, 7th and 21st day after the treatment. While selamectin was 100% effective in removing M. musculi, M. musculinus and R. ensifera by the seventh day, its effect against S. obvelata and A. tetraptera was 36.7% and 49.2%, respectively on the 21st day.     

Trypanosoma musculi with Trichinella spiralis or Heligmosomoides polygyrus: concomitant infections in the mouse

Inbred mice infected with Trypanosoma musculi displayed wide variations in peak blood parasitemia. The most susceptible mice were C3H and A strain, while Balb/c, C57B1/6, and the related congenic B10 strains were the most resistant. The effect of an intestinal infection with either Trichinella spiralis or Heligmosomoides polygyrus on proliferation of T. musculi was investigated. T. spiralis infections given at the same time or up to 45 days before a T. musculi infection always caused an increase in blood parasitemia in C3H mice. Maximum increases were observed when T. spiralis infections preceded T. musculi by 5-10 days. In all mouse strains examined, dual infections increased maximum parasitemia by two- to four-fold, regardless of the degree of resistance of that mouse strain to either T. musculi or T. spiralis. This suggested that the immunological "cost" of a T. spiralis infection was the same for strains that were strong or weak responders to a primary infection with T. spiralis. In contrast, infection with H. polygyrus did not promote T. musculi parasitemia over the level of a single infection. The increase in blood parasitemia in T. spiralis-infected mice was largely due to the intestinal adult worm, but migratory larvae and mature muscle larvae also stimulated increased parasitemias. The increase in parasitemia was proportionate to the dose of T. spiralis, and the sex of the host did not affect the blood trypanosome level.     

Evaluation of the control of Myobia musculi infestations on laboratory mice with permethrin

The efficacy of permethrin against Myobia musculi (Schrank) (Acari:Myobiidae) infestations on mice was evaluated using four different dose delivery methods. In all methods, an attempt was made to deliver 0.5 mg of active permethrin weekly to each mouse. Successful control was achieved with topical or environmental treatment with 0.25% permethrin dust and by dipping the mice into a 0.6 g/l permethrin emulsion. The use of an emulsion applied to the bedding produced the least satisfactory control. Untreated mice had an average infestation rate of 57.3% throughout the study. No significant differences were seen between treated and untreated groups in either body weight or histopathology of the liver, lung, or kidney.     

Effect of malnutrition on susceptibility of mice to Trypanosoma musculi: vitamin A-deficiency.

Vitamin A-deficiency was studied in mice infected with Trypanosoma musculi. Irrespective of diet, trypomastigotes (trypanosomes) appeared in peripheral tail blood of all inoculated mice after 6-day incubation periods. On the average, vitamin A-deficient mice had parasitemias about 10 times greater than animals fed a complete diet and 8 times pair-fed controls. Parasitemias lasted longer in vitamin-deficient hosts, and reached a maximum five days later than those from control hosts.     

Macrophage cytostatic effect on Trypanosoma musculi involves an L-arginine-dependent mechanism

Peritoneal macrophages from mice infected with an extracellular parasite, Trypanosoma musculi were effective in inhibiting parasite proliferation in vitro. This trypanostatic activity could be suppressed by NG monomethyl-L-arginine (NGMMA), a specific inhibitor of a biochemical pathway synthesizing L-citrulline and inorganic nitrogen oxides from L-arginine. Macrophages exerted this in vitro antiproliferative effect from the 10th day of infection on and this activity was maximum around 14th day of infection. Nitrite production paralleled development of macrophage trypanostatic activity. Macrophages collected from BCG-infected mice or treated with IFN-gamma in vitro also exerted a trypanostatic activity which was suppressed by NGMMA. A trypanostatic activity suppressed by NGMMA was also exerted by splenic macrophages from T. musculi-infected mice. Trypanostatic activity of IFN-gamma-treated macrophages was reduced by addition of anti-TNF-alpha showing the participation of TNF-alpha in IFN-gamma-mediated macrophage trypanostatic activity. Nitric oxide (NO) gas inhibited T. musculi proliferation. Addition of excess iron reversed the trypanostatic effect of both macrophages and NO gas. All these data showed that, as reported for a broad spectrum of microorganisms, activated macrophages displayed an antimicrobial effect on trypanosomes through the L-arginine: NO pathway that could participate in controlling infection in T. musculi-infected mice before appearance of antibody-dependent mechanisms. NO production by activated macrophages could trigger iron loss from critical target enzymes in trypanosomes.     

Recovery from immunesuppression in mice infected with Schistosoma japonicum by treatment of praziquantel

To learn the possible alteration of immune response, hemolytic plaque-forming cells (PFC) produced in the spleen of Schistosoma japonicum infected mice treated with Praziquantel and untreated group were counted. There was no significant difference in the immunesuppression percentage between the treated and untreated groups 1 and 3 weeks after treatment. In 5 weeks after treatment, however, the immunesuppression percentage in the treated mice was markedly reduced in comparison with that of the untreated group. Recovery from immunesuppression appears to be associated with elimination or impairment of adult worms.     

The pathogenesis of trehalose dimycolate-induced interstitial pneumonitis: III. Evidence for a role for T lymphocytes

Trehalose dimycolate, a glycolipid component of the cell walls of mycobacteria, induces interstitial pneumonitis and alveolar hemorrhages in C57BL/6 and C57BL/10 mice. Homozygous nude (nu/nu) mice of these backgrounds are not susceptible to this form of pulmonary injury. However, after administration of T-lymphocyte-enriched spleen cell preparations from syngeneic donors, homozygous nude mice become susceptible to trehalose dimycolate. The observations suggest that production of pulmonary lesions by this mycobacterial component is dependent on T lymphocytes. While the mechanisms are still under study, we propose that trehalose dimycolate can function as an activator of T lymphocytes and that products of activated T cells are responsible for production of the pulmonary lesions.     

Arthropod transmission of Eperythrozoon coccoides in mice

The association of Eperythrozoon coccoides infection in laboratory mice, with commonly encountered external parasites, was examined. Transmission of E. coccoides was achieved by infesting recipient mice with as few as 10 live Polyplax serrata immediately after they were obtained from donor mice infected with E. coccoides. Polyplax serrata removed from such hosts and fasted for 24 hours also were able to initiate E. coccoides infections in recipient mice. Transovarial transmission of E. coccoides through P. serrata was not demonstrated. Transmission of E. coccoides by Myocoptes musculinus, Myobia musculi and Radfordia affinis was not observed.     

Stimulation of the cellular immune response to lymphocytic choriomeningitis (LCM) virus infection in suckling mice

Death occurred earlier and its rate was higher in one-week-old mice treated with phytohaemagglutinin (PHA) and subsequently inoculated intracerebrally with LCM virus than in their virus infected but untreated littermates. Thus PHA treatment contributed to the outcome of LCM virus infection in the form of lethal meningitis. The course of LCM virus infection in 1-week-old PHA treated mice was similar as in the untreated 2-week-old mice. This indicates that PHA treatment accelerated the development of cell mediated immunological capacity in suckling mice.     

The histopathological and immunological pattern of CBA mice infected with Leishmania amazonensis after treatment with pyrazole carbohydrazide derivatives

Because there is no vaccine in clinical use, control of Leishmaniasis relies almost exclusively on chemotherapy and the conventional treatments exhibit high toxicity for patients and emerging drug resistance. Recently, we showed that oral treatment with synthetic pyrazole carbohydrazide compounds induced lower parasite load in draining lymph nodes and reduced skin lesion size without causing any toxic effects in an experimental murine infection model with Leishmania amazonensis. In this study, CBA mice were infected in the footpad with L. amazonensis and then orally treated with pyrazole carbohydrazides derivatives, such as BrNO₂, NO₂Cl and NO₂Br and their histopathological and immunological effects were then investigated. Epidermis and dermis had lower levels of inflammatory infiltration compared to the infected untreated control mice. In the dermis of treated animals, the numbers of vacuolated macrophages containing intracellular parasites were far lower than in infected untreated animals. In addition to dermal macrophages, we also observed a mixed inflammatory infiltrate containing lymphocytes and granulocyte cells. Lower numbers of B cells (B220+) and T lymphocytes (CD3+) were identified in the lesions of treated mice compared with the untreated, infected mice. In draining lymph node cells, the number of T lymphocytes (CD3+) was decreased, and the numbers of B cells (CD19+) and CD8+ T cells were increased in infected mice, when compared with the non-infected control group. In additional, we have shown that infected treated and untreated lymph node cells had similar levels of TGF-β and IFN-γ mRNA expression, whereas IL-4 was expressed at a lower level in the treated group. Increased levels of the specific anti-Leishmania IgG2a or IgG3 antibody subclass were observed in NO₂Cl or BrNO₂-treated group, respectively. Overall, our experimental findings suggest that pyrazole carbohydrazides exert modulation of IL-4 expression and B cell levels; however, further evaluation is required to determine the optimal treatment regime.     

Increase in S-adenosylmethionine decarboxylase in SV-3T3 cells treated with S-methyl-5'-methylthioadenosine.

We previously have shown [Takahashi & Kobayashi (1982) Hepatology 2, 249-254] that the administration of concanavalin A to mice with schistosomiasis caused liver collagen content to be reduced by 50%. Here we report the effects of concanavalin A and aggregated mouse myeloma IgG on liver lysyl oxidase activity and present further evidence concerning the possible mechanism by which the liver collagen content was decreased in infected-treated mice. The lysyl oxidase activity at 8 weeks after infection in both treated mice and untreated infected controls was about 28-fold greater than in the age-matched uninfected controls. The specific radioactivity of intracellular free [14C]proline, the rate of collagen synthesis, the ratio of collagenase-sensitive, protein-bound, hydroxyproline to proline of collagen and the intracellular degradation of newly synthesized collagen were similar in treated animals and in untreated infected controls. In contrast, the extracellular degradation of newly secreted collagen and the specific radioactivity of protein-bound [14C]hydroxyproline in the agent-treated groups were about 2-fold greater than those in the untreated infected controls. These results suggest that the observed 50% decrease in content of liver collagen of mice treated with the agents apparently was due to the increased extracellular degradation of newly secreted collagen.     

In vivo activity of released cell wall lipids of Mycobacterium bovis bacillus Calmette-Guérin is due principally to trehalose mycolates

The hallmark of Mycobacterium-induced pathology is granulomatous inflammation at the site of infection. Mycobacterial lipids are potent immunomodulators that contribute to the granulomatous response and are released in appreciable quantities by intracellular bacilli. Previously we investigated the granulomagenic nature of the peripheral cell wall lipids of Mycobacterium bovis bacillus Calmette-Guérin (BCG) by coating the lipids onto 90-microm diameter microspheres that were mixed into Matrigel matrix with syngeneic bone marrow-derived macrophages and injected i.p. into mice. These studies demonstrated that BCG lipids elicit proinflammatory cytokines and recruit leukocytes. In the current study we determined the lipids responsible for this proinflammatory effect. BCG-derived cell wall lipids were fractionated and purified by liquid chromatography and preparative TLC. The isolated fractions including phosphatidylinositol dimannosides, cardiolipin, phosphatidylglycerol, phosphatidylethanolamine, trehalose monomycolate, trehalose dimycolate, and mycoside B. Trehalose dimycolate, when delivered to bone marrow-derived murine macrophages, induced the greatest secretion of IL-1beta, IL-6, and TNF-alpha in vitro. Trehalose dimycolate similarly induced the greatest secretion of these proinflammatory cytokines in ex vivo matrices over the course of 12 days. Trehalose monomycolate and dimycolate also induced profound neutrophil recruitment in vivo. Experiments with TLR2 or TLR4 gene-deficient mice revealed no defects in responses to trehalose mycolates, although MyD88-deficient mice manifested significantly reduced cell recruitment and cytokine production. These results demonstrate that the trehalose mycolates, particularly trehalose dimycolate, are the most bioactive lipids in the BCG extract, inducing a proinflammatory cascade that influences granuloma formation.     

Trypanosoma musculi: population dynamics of erythrocytes and leukocytes during the course of murine infections

Cells of the hemocytic and lymphoreticular series located in the blood, bone marrow, spleen, and peritoneal space have been analyzed throughout the course of Trypanosoma musculi infections of intact and splenectomized C3H female mice. Following an early (within 2 days after trypanosome inoculation intraperitoneally) shift of leukocytes from the blood to the peritoneal space, there occurred a more gradual, prolonged infusion of leukocytes into the peritoneal space, the primary site of infection, that continued until the infection was terminated. There was intense cytogeneractive activity in the spleen that included erythrocytes, lymphocytes, myelocytes, and megakaryocytes. The marrow became primarily a site of monocytopoiesis and, to some extent, of lymphopoiesis. During the first 8 days (approximately) of infection, there was a decline in mature erythrocytes in the blood (the well-known anemia) and development of a profound thrombocytopenia. In splenectomized mice, the depletion of these elements continued unabated until the mice died; the marrow of infected, splenectomized mice failed to provide these elements, as was also the case in intact mice. In the peritoneal space, the intense battle between leukocytes and trypanosomes was reflected in a gradual, impressive rise in the number of dead and fatigued cells and, late in infection, in the development of ascites. Both of these abnormal conditions disappeared shortly after cure of the infection. We conclude that infections of mice with T. musculi result in dedication of the entire lymphoreticular system to the generation of cells that are exported to the peritoneal space to combat the major infection the occurs in that locale. This is consistent with the evidence that the belated immune elimination of T. musculi is a cell-mediated (probably antibody-dependent) process. The disruption of the normal histoarchitecture, the shift in the normal proportions of cells and in cells of different degrees of maturity, and probably, a block imposed on precursor cell maturation, account to a large extent for the well-known failure of immune responses commonly associated with trypanosome infections.     

Reversibility of cardiac fibrosis in mice chronically infected with Trypanosoma cruzi, under specific chemotherapy.

This investigation was performed to verify the effect of specific chemotherapy (Benznidazole or MK-436) on the inflammatory and fibrotic cardiac alterations in mice chronically infected with the strains 21 SF (Type II) and Colombian (Type III) of Trypanosoma cruzi. To obtain chronically infected mice, two groups of 100 Swiss mice each, were infected with either the 21 SF or the Colombian strain (2 x 10(4) and 5 x 10(4) blood forms respectively). The rate of mortality in the acute phase was of 80% for both groups. Twenty surviving mice chronically infected with the 21 SF strain and 20 with the Colombian strain were then divided in treated and untreated groups. Excluding those that died during the course of treatment, 14 mice chronically infected with the 21 SF strain and 15 with the Colombian strain were finally evaluated in the present study. Chemotherapy was performed with Benznidazole (N-benzil-2-nitro-1-imidazolacetamide) in the dose of 100 mg/k.b.w/day, for 60 days, or with the MK-436 (3(1-methyl-5 nitroimidazol-2-yl) in two daily doses of 250 mg/k.b.w, for 20 days. Parasitological cure tests were performed (xenodiagnosis, haemoculture, subinoculation of the blood into newborn mice), and serological indirect immunofluorescence test. The treated and untreated mice as well as intact controls were killed at different periods after treatment and the heart were submitted to histopathological study with hematoxilineosin and picrosirius staining; ultrastructural study; collagen immunotyping, fibronectin and laminin identification by immunofluorescence tests. Results: the untreated controls either infected with 21 SF or Colombian strain, showed inflammatory and fibrotic alterations that were mild to moderate with the 21 SF strain and intense with the Colombian strain. Redpicrosirius staining showed bundles of collagen in the interstitial space and around cardiac fibers. Increased deposits of matritial components and collagen fibers, macrophages and fibroblasts appeared at the ultrastructural examination. Deposits of fibronectin, laminin, pro-III and IV collagens were seen, most intense in those infected with the Colombian strain. Treated mice, parasitologically cured, presented clear-cut regression of the inflammatory lesions and of the interstitial matrix thickening. Mice infected with the Colombian strain and treated with MK-436, was parasitologically cured in 5/6 cases and showed mild inflammatory infiltration and fibrosis. The mice treated with Benznidazole (Colombian strain) did not cure and showed moderate fibrosis and inflammation. Treatment of the mice infected with the 21 SF with Benznidazole determined parasitological cure of all animals, that showed mild inflammation and fibrosis of the myocardium.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cord factor (trehalose-6-6'-dimycolate) of in vivo-derived Mycobacterium lepraemurium.

Harvests of Mycobacterium lepraemurium obtained from livers of moribund infected mice yielded M. lepraemurium cell walls that were extracted with solvent to provide crude M. lepraemurium cell wall lipids. By solvent fractionation and chromatography on DEAE cellulose and cellulose, a cord factor-like glycolipids contaminated with mycoside C was obtained. Additional solvent treatment provided the purified glycolipid, which was identified as 6,6'-trehalose dimycolate, by infrared and chromatographic comparison with authentic samples from M. tuberculosis, by identification of trehalose and specific mycolates of M. lepraemurium, and by permethylation analysis. This constitutes the first unequivocal identification of cord factor as a product of in vivo-derived mycobacteria.     

The activity of nifluridide on reduviids and rodent and human trypanosomes

The ability of nifluridide to kill reduviids was assayed in mice fed 7 ppm in diet and on cattle injected subcutaneously at 5 mg/kg body weight. Nifluridide was systemically active against Triatoma infestans on mice and Rhodnius prolixus on cattle. No effects on Trypanosoma (Schizotrypanum) cruzi could be detected in the intestinal contents of Triatoma infestans killed by the compound. In vitro and in vivo studies were conducted to determine the effects of nifluridide on trypanosomes growing in medium and in experimentally infected mice. Culture forms of Trypanosoma cruzi grown at 27 degrees C that are morphologically similar to epimastigotes found in infected bugs were affected by 2.5 to 10 ppm in the medium. Mice fed nifluridide in the diet simultaneous with infection of Trypanosoma cruzi or Trypanosoma (Herpetosoma) musculi exhibited parasitemias and tissue infections similar to nontreated infected mice. At the concentration tested, bloodstream trypomastigotes and culture epimastigotes of Trypanosoma musculi were unaffected by nifluridide. Only the culture epimastigotes of Trypanosoma cruzi were affected by the drug but not the bloodstream and tissue forms.     

Regulation of inflammatory responses to Bordetella pertussis by N(G)-monomethyl-L-arginine in mice intranasally infected

To investigate effect of MMLA, an inhibitor of nitric oxide (NO) production, on regulation of inflammatory responses to Bordetella pertussis infection, mice were infected intranasally, and treated with various concentrations of MMLA. Ten days after infection, mice treated with MMLA at dosage of 100 mg/kg, given intraperitoneally in a single dose or for 5 consecutive days, showed at histopathologic examination, a significant decrease of intensity of inflammation (scores, 0.6 +/- 0.2 and 0.9 +/- 0.5 respectively). A decrease of cellular accumulation of neutrophils and lymphocytes in the bronchoalveolar lavage (BAL) fluid was observed in infected mice treated with MMLA, especially at dosage of 10 mg/kg, given in a single dose intraperitoneally. In addition, BP-infected mice treated with MMLA (100 mg/kg, intraperitoneally) for 5 consecutive days showed higher mortality rate than untreated mice infected with B. pertussis, and the number of B. pertussis in lungs of mice treated with MMLA was significantly increased. However, MMLA treatment of infected mice had some effect on levels of IFN-gamma and nitrite/nitrate (end-stable products of NO) in the BAL fluid. This study indicates that NO may play a role either as microbiocidal agent or as a modulator of immune regulation, inasmuch as it may upregulate tissue inflammatory response to B. pertussis.     

Trehalose dimycolate enhances survival of fission neutron-irradiated mice and Klebsiella pneumoniae-challenged irradiated mice

The survival of B6D2F1 female mice exposed to lethal doses of fission neutron radiation is increased when trehalose dimycolate (TDM) preparations are given either 1 h after exposure or 1 day before exposure to radiation. TDM in an emulsion of squalene, Tween 80, and saline was the most effective formulation for increasing the 30-day survival of mice when given 1 day before (90%) or 1 h after (88%) exposure to radiation. An aqueous suspension of a synthetic analog of TDM was less effective at increasing 30-day survival (60%) when given 1 day prior to radiation exposure and not effective when given 1 h after radiation. Mice receiving a sublethal dose (3.5 Gy) of fission neutron radiation and either the TDM emulsion or synthetic TDM 1 h after irradiation were substantially more resistant to challenge with 10, 100, 1000, or 5000 times the LD50/30 dose of Klebsiella pneumoniae than untreated mice.