Trypanocidal drugs and the role of kidney forms of Trypanosoma (Herpetosoma) musculi in immunity of mice against reinfection

Adrenals, hearts, kidneys, livers, lungs, and spleens were removed from C3H/Anf mice which had been inoculated with Trypanosoma (Herpetosoma) musculi and no longer exhibited parasitemias. Imprints of each organ were examined microscopically, and each was homogenized and injected into recipient mice. It was confirmed that trypanosomes could be detected only in the donor kidneys. Lampit or Ethidium treatment eliminated bloodstream and kidney forms when administration was initiated after the development of patent parasitemias. However, mice treated with Lampit on the same day they were inoculated with T. musculi developed parasitemias later than animals injected with drug after parasites had appeared in their blood. Both Lampit and Ethidium depressed antibody production as detected in enzyme-linked immunosorbent assays of antisera from animals having parasitemias at the time of treatment. The elimination of kidney forms by Lampit or Ethidium treatment did not reduce the resistance of mice to reinfection by T. musculi 12 weeks or 15 and 22 weeks, respectively, after the initial inoculation of these animals with the parasites. Kidney forms were not required for the sustained protective immunity of the mice against reinfection during the intervals of these experiments.     

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.     

Adoptive cell transfer studies to examine the role of T lymphocytes in immunity to Trypanosoma musculi

Previous studies in this laboratory utilizing monoclonal antibody-induced immunosuppression have demonstrated that the T-helper lymphocyte is primarily responsible for the T lymphocyte dependency of Trypanosoma musculi elimination from the bloodstream of mice, and that T-cytotoxic lymphocytes play a minimal role in this response. In the current study, these findings were extended by examining the effects of adoptive cell transfers on the course of infection with T. musculi using immune splenocytes enriched for T lymphocyte subpopulations. These studies demonstrated that adoptive transfer of immune splenic T lymphocytes resulted in a specific, dose-related enhancement of kinetics of trypanosome elimination. This effect was found to be due to the presence of L3T4+ T-helper cells in the immune splenocyte population. Adoptive transfer of Lyt-2+ T-cytotoxic cells or lymphokine-activated killer (LAK) cells was ineffective in altering the course of infection. In addition, it was found that immune B lymphocytes were equally capable of adoptively transferring immunity to T. musculi, suggesting that the primary role of the T-helper lymphocyte is to provide help in the induction of parasite-specific antibodies.     

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.     

The kidney form of Trypanosoma musculi: A distinct stage in the life cycle?

Trypanosoma musculi is a parasite specific to mice, which resides in the blood and lacks intracellular stages. After immune clearance of the flagellates from the general circulation, mice are resistant to reinfection. Yet, long after parasites are no longer detected in the peripheral blood, they persist in the vasa recta of the kidneys and it has been proposed that this is an immunologically privileged site for T. musculi. This relationship provides a useful model for studies of latent or chronic infections in immune hosts. Here, Fernando Monroy and Donald Dusanic consider the immune responses of mice to T. musculi and compare characteristics of the parasites from the vasa recta (kidney forms, KFs) of mice with latent infections to trypanosomes from the peripheral blood (bloodstream forms, BSFs) of animals during active infections. They consider how KFs evade immune destruction and suggest that these sequestered parasites represent a distinct stage in the life cycle.     

Trypanosoma musculi: characterization of the T-lymphocyte dependency of immunity by selective immunomodulation of the mouse, Mus musculus

Trypanosoma musculi is a hemoflagellate parasite producing a characteristic course of infection in the mouse, terminating in elimination of the parasite from the vascular system with permanent immunity to reinfection. While this elimination is known to require functional T lymphocytes, the role of these cells has remained undefined. The current study examined the role of T-lymphocyte dependency by the selective in vivo depletion of T-lymphocyte subsets by monoclonal antibodies or immunosuppressive pharmacologic agents. In the former approach, monoclonal antibodies specific for the murine T-lymphocyte surface antigens Thy 1.2, Lyt 2, or L3T4 were administered to mice, resulting in functional depletion of either all T lymphocytes or the T-helper or T-cytotoxic subsets. Subsequent infection with T. musculi revealed that elimination of the T-helper, but not the T-cytotoxic, phenotype resulted in a prolonged and elevated parasitemia. This enhancement of parasitemia was similar to that produced with depletion of all T lymphocytes (i.e., Thy 1.2+ cells), suggesting that the T-helper cell was most important in parasite elimination. In the latter approach, pharmacologic agents displaying selective immunosuppressive activity were employed to inactivate, rather than destroy, T-lymphocyte subpopulations. In vivo inactivation of T-helper lymphocytes by the fungal metabolite cyclosporin-A enhanced the level of parasitemia in treated animals and resulted in a protracted duration of infection. In contrast, treatment with the highly substituted anthracenedione AEAD, which has been demonstrated to selectively inactivate T-cytotoxic cells, had no appreciable effect on the kinetics of infection. These results strongly suggest that the T-lymphocyte dependency of Trypanosoma musculi elimination is due predominantly to the action of the T-helper, rather than T-cytotoxic, lymphocyte.     

海蟾夏眠的代谢生理研究

许多两栖的脊椎动物在干早缺水或者食物不足时埋藏在泥土里休眠。例如,肺鱼能在泥茧里不取食与饮水而生存数年之久(Delaney等,1977),生活在沙漠地带的两栖类能埋藏在沙土里数月以躲避干旱,海蟾(Bufo marinus)能埋藏在沙里克服短时间的缺水,而以露在空气中的鼠孔进行呼吸(Boutilier等,1979)。 海蟾在失水时,经皮肤排出的二氧化碳减少;但是,失水而尚未埋藏在沙里的海蟾,     

An endoplasmic reticulum-specific cyclophilin.

Cyclophilin is a ubiquitously expressed cytosolic peptidyl-prolyl cis-trans isomerase that is inhibited by the immunosuppressive drug cyclosporin A. A degenerate oligonucleotide based on a conserved cyclophilin sequence was used to isolate cDNA clones representing a ubiquitously expressed mRNA from mice and humans. This mRNA encodes a novel 20-kDa protein, CPH2, that shares 64% sequence identity with cyclophilin. Bacterially expressed CPH2 binds cyclosporin A and is a cyclosporin A-inhibitable peptidyl-prolyl cis-trans isomerase. Cell fractionation of rat liver followed by Western blot (immunoblot) analysis indicated that CPH2 is not cytosolic but rather is located exclusively in the endoplasmic reticulum. These results suggest that cyclosporin A mediates its effect on cells through more than one cyclophilin and that cyclosporin A-induced misfolding of T-cell membrane proteins normally mediated by CPH2 plays a role in immunosuppression.     

寒冷刺激对部分睡眠剥夺小鼠血细胞参数的影响

目的：探讨寒冷刺激,部分睡眠剥夺,以及部分睡眠剥夺的基础上再给予寒冷刺激等处理对小鼠血细胞参数的影响。方法：昆明小鼠24只,随机均分为4组（n=6）：对照组、寒冷组、不完全睡眠剥夺组和不完全睡眠剥夺加寒冷组。寒冷组每天给予（10±2）℃的低温处理4h,不完全睡眠剥夺组每天18：00至次日9：00剥夺睡眠,不完全睡眠剥夺加寒冷组在每天睡眠剥夺的基础上再给予4h寒冷刺激。连续处理4d后采血检测血常规和血沉率。结果：与对照组相比,寒冷刺激可使小鼠血液中淋巴细胞含量（P〈0．05）以及百分比（P〈0．01）显著增加;部分睡眠剥夺可使小鼠血液白细胞和淋巴细胞含量（P〈0．05）及百分比（P〈0．01）明显降低。不完全睡眠剥夺加寒冷刺激处理后与其它三组相比小鼠血液白细胞和淋巴细胞含量显著降低（P〈0．01）,而血沉率则显著升高（P〈0．01）。结论：部分睡眠剥夺会抑制机体免疫能力,在部分剥夺睡眠的基础上再给予寒冷刺激则将进一步抑制机体免疫能力并使血沉率加快,降低机体对外界环境变化的适应能力。     

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.     

Protective effect of trehalose dimycolate in infestation of mice by Trypanosoma musculi

Trehalose dimycolate (TDM) treated and untreated mice were infected with Trypanosoma musculi. Compared to untreated mice, treated mice exhibited a five fold reduced number of circulating parasites. Untreated infected mice had a splenomegaly but only a slight increase of spleen weight of treated mice was observed. The role of trehalose dimycolate on T. musculi infection, especially via the macrophage is discussed.     

The nature of immunodeficiency induced by injections of lectins and cyclophosphamide

Consecutive injections of T-cell mitogen (LcA, Con A) and cyclophosphamide (CY) produce an inhibition of T-cell, but not B-cell functions. This phenomenon was not a result of suppressor-cell activity of the action of some suppressor serum factors. Immunoreactivity of mice, treated with Con A CY is restored by thymocytes from intact donors, but not bone marrow cells. Using, monoclonal antibodies to Thy-1.2 antigen or anti-Ig serum it has been shown that pretreatment of mice with lectin and CY resulted in a decrease in T-cell, but not B-cell number in comparison with control mice. The above facts are indicative of CY-mediated elimination of T cells involved in proliferation and differentiation by lectin.     

Partial hepatectomy aggravates cyclosporin A-induced neurotoxicity by lowering the function of the blood-brain barrier in mice

AimsCyclosporin A, a calcineurin inhibitor, produces neurotoxicity with relatively high frequency in organ-transplanted patients. The aim of the present study was to clarify whether acute liver failure (ALF) simulated to the transient liver dysfunction at an early phase after liver transplantation increases the susceptibility to cyclosporin A-induced neurotoxicity through the blood–brain barrier (BBB) dysfunction.Main methodsThe right internal, left lateral and left internal lobes in male ddy mice were surgically excised under sodium pentobarbital anesthesia. Effect of cyclosporin A on harmine-induced tremors was examined and BBB permeability to ³[H]cyclosporin A was assessed in partially (70%) hepatectomized mice at postoperative days 1, 3 and 7.Key findingsPatrial hepatectomy aggravated harmine-induced tremors. Cyclosporin A (50 mg/kg, i.p.) markedly augmented harmine-induced tremors in partially hepatectomized mice at postoperative day 1. Consistent with these behavioral findings, the brain uptake of ³[H]cyclosporin A in mice injected with ³[H]cyclosporin A into the jugular vein at postoperative day 1 was significantly increased by partial hepatectomy compared with sham operation.SignificanceOur results indicate that ALF increases BBB permeability to cyclosporin A by lowering the function of P-glycoprotein and tight junctions, consequently leading to augmentation of cyclosporin A-induced neurotoxicity. The possibility that cyclosporin A increases the risk of neurotoxicity including tremors at an early phase of liver transplantation must be considered.     

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.     

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.     

Diminished reproduction, failure to thrive, and altered immunologic function in a colony of T-cell receptor transgenic mice: possible role of Citrobacter rodentium

Citrobacter rodentium from an undetermined source was detected in a breeding colony of T-cell receptor transgenic mice housed in a conventional mouse facility in which murine hepatitis virus had been endemic and Helicobacter spp. had been detected. Citrobacter rodentium, isolated from blood, spleen, and colon, correlated with a significant increase in mortality and morbidity in this breeding colony. Transgenic mice of all ages were affected by chronic debilitation, loss in reproductive efficiency, rectal prolapse, and acute death, resulting in the near loss of these noncommercially available strains. Several alterations in immunologic parameters were observed, including outgrowth of an unusual population of cells in the spleen and blood, reduction in ascites production, loss of the capacity of peritoneal exudate cells to serve as feeders for the cloning of long-term T-cell lines, and inhibition of antigen-specific cytotoxic T-cell activity. These altered immune functions also were apparent in commercially-derived nontransgenic mice cohoused with the infected colony and in overtly healthy transgenic and nontransgenic littermates. Citrobacter rodentium and murine hepatitis virus were eliminated ultimately on rederivation of the affected strains by embryo transfer. However, the rapid decrease in the health of the colony necessitated more immediate action. To reduce mortality and allow breeding to continue during rederivation of the transgenic lines, animals were treated with enrofloxacin and moved to a barrier facility. Antibiotic therapy significantly reduced morbidity and mortality, markedly increased litter size and frequency, and resulted in the normalization of many of the immunologic assays. The involvement of C. rodentium in altering viability of the colony and perturbing immunologic assays is suggested by correlation of the onset of the syndrome with the appearance of Citrobacter sp. and its resolution with the elimination of Citrobacter sp. from the colony. Whether infection with Citrobacter alone is causative or whether superinfection of murine hepatitis virus- and Helicobacter-infected mice is required remains to be determined.     

The effect of food and water deprivation on the peripheral blood parameters of the mouse

Various peripheral blood and bone marrow parameters were determined during food and water deprivation and during food deprivation alone in order to obtain base lines that may be used to make comparisons with similar data from irradiated mice. The peripheral blood parameters following food and water deprivation were similar to those following food deprivation alone. The mean survival time was about 5 days and the weight loss 40% of the control weight. There was an absolute decrease in the total circulating lymphocyte and platelet counts, while the total granulocyte count remained unchanged or increased. The blood volume decreased, while the hematocrit and specific gravity of the blood increased. The bone marrow parameters following food and water deprivation showed that erythropoiesis was more markedly depressed than myelopoiesis. The tritiated thymidine labeling index for granulopoietic cells and megakaryocytes decreased progressively during starvation. The variations in the white blood count and the bone marrow parameters are not comparable with those found in irradiated mice having the G.I. syndrome; the changes in mean survival time, weight loss, hematocrit, and blood volume are similar.     

Aging of the murine immune system is reflected by declining ability to generate antibodies that promote elimination of Trypanosoma musculi

Trypanosoma musculi established extracellular infections in aged BC3F1 and C57BL/6 mice that were approximately 10 times greater and twice as long in duration as those in young adult mice. Elimination of T. musculi infections was found to be an antibody-dependent, cell-mediated process involving effector (presumably phagocytic) cells in the liver and, to a lesser extent, the spleen. A major difference between young and aged mice of the C57BL/6 strain was the deficiency in the ability of aged animals to generate antibodies of appropriate specificity and/or isotype in sufficient amount to promote trypanosome elimination. Indeed, at the time when infected young-adult mice began to produce antibodies that facilitated rapid trypanosome clearance (in young adult, but not in aged animals), the serum of aged mice was found to contain substances that inhibited parasite clearance. Overall, however, the development of antibodies of different isotypes, capable of reacting with intact trypanosomes, was about the same in young and aged animals. Hepatic and splenic effector cells of old mice were at least as efficient as those of young adults. The immunoblotting procedure was used to try to detect trypanosome Ag against which aged animals failed to generate antibodies of one or more isotypes. The complexity of the reactions of infected mouse serum antibodies with the spectrum of trypanosome Ag precluded a precise analysis. However, it was apparent that a delay in the appearance of antibodies of IgG2a and IgG2b isotypes, against Ag of relatively high m.w., was typical of aged, in comparison to young, mice. More exacting analyses, involving fractionated trypanosome extracts and mAb of varying isotypes, are underway to identify key trypanosome Ag that elicit antibodies of isotypes that can facilitate hepatic and splenic clearance of the parasites. This line of investigation will provide information that, in addition to its intrinsic interest, may be vital in judging the future impact of endemic parasitic infections on the emerging cohort of elderly persons in developing tropical nations.     

Characterization of Cells Infiltrating the Lungs of X-Irradiated and Nude Mice after Influenza Virus Infection

After X-irradiated and nonirradiated mice (C3H/He) as well as athymic nude mice and haired littermates (BALB/c) were infected with influenza A virus (Kumamoto strain, H2N2), they were examined for survival period, the development of consolidation in the lungs and the characteristics of the cells infiltrating the lung tissues. In two different T-cell deficient groups, there was a definite delay in the development of consolidation compared with their respective controls and this was reflected in prolonged survival periods: 5 days longer for irradiated mice and 6 days longer for nude mice. In both T-cell deficient and normal groups, about 70% of the cells obtained from consolidated lung tissues after virus infection were found to be small lymphoid cells and there were no morphological differences between the T-cell deficient and normal groups. None of these small lymphoid cells from the peripheral blood or the spleens of T-cell deficient mice responded to concanavalin A. In the lungs of both X-irradiated mice and nude mice, however, a definite increase in cells having natural killer activity was found at the late stages of the influenza infection, suggesting their participation in the development of consolidation.     

Enhanced Engraftment of HTLV-I-Infected Human T Cells in Severe Combined Immunodeficiency Mice by Anti-asialo GM-1 Antibody Treatment

The effects of anti-asialo GM-1 antibody (AAGM) treatment on the engraftment of human T-cell leukemia virus type I (HTLV-I)-infected human T cells in severe combined immunodeficiency (SCID) mice were studied. The frequency of tumor formation in an HTLV-I-transformed human T-cell line, MT-2 cells, at the site of inoculation was significantly higher in AAGM-treated than untreated mice (P<0.05): 16/18 (89%) and 16/26 (62%), respectively. The promotive effect of AAGM treatment on tumor development was marked in the early stage (less than 3 weeks), suggesting that the immediate reaction of natural killers to the inoculated cells may be important for the prevention of tumor development. The surface phenotypes and clonality of the tumor cells were the same as the MT-2 cells inoculated. Inoculation of peripheral blood mononuclear cells (PBMC) from one of the 4 adult T-cell leukemia/lymphoma (ATL) patients resulted in the development of tumors in AAGM-treated SCID mice. However, the surface phenotypes of the cells from these tumors were a mixture of B cells and T cells, suggesting that these tumors consisted of Epstein-Barr virus-transformed B cells and HTLV-I-transformed T cells. In addition, HTLV-I was detected by polymerase chain reaction in various organs of the mice inoculated with PBMC from the ATL patient and the asymptomatic carrier examined. These results suggest that elimination of natural killer function by AAGM treatment is important, although such treatment is not always necessary for the engraftment of HTLV-I-infected cells in SCID mice.