Cellular immune responses of jirds to extracts of life cycle stages and adult excretory secretory products during the early development of Brugia pahangi

The Brugia-jird model of lymphatic filariasis was used to examine the induction of cellular immune responses during the early premicrofilaremic phases of the infection. The intensity of the pulmonary granulomatous inflammatory response (PGRN) was determined by measuring granuloma areas around Sepharose beads coated with parasite extracts which were embolized in the lungs of jirds prior to necropsy. Necropsies were performed at 7, 14, 28, 56, and 150 days postinfection (DPI). These time points correspond to specific developmental changes in the life cycle. Lymphocyte blastogenesis assays were performed using cells from draining renal lymph nodes and splenocytes at 14 and 150 DPI. Soluble extracts of third stage larvae (L3), fourth stage larvae (L4), adult females, adult males, microfilariae (MF), and excretory secretory products (ES) of males and females were used in both measurements of cellular responsiveness. A marked granulomatous response to parasite extracts peaked at 7 DPI or 14 DPI followed by a gradual decrease to a hyporesponsive state at 120 DPI. The response of renal lymph node cells also was significantly elevated at 14 DPI and significantly decreased at > 150 DPI. The splenocyte responses were erratic and did not follow this pattern. Significant differences in PGRN responses to somatic extract preparations were not seen during the early stages of the infection (7, 14, 28 DPI), but those to MF and L3 were significantly less at 56 and 120 DPI. Although PGRN responses to ES followed a similar pattern, these were less than those to the somatic extract. The data indicated that a rapid, intense cell-mediated inflammatory response is induced early during a primary infection and that this response is rapidly downregulated. This downregulation begins prior to the maturation of adult parasites and microfilarial production. The early phase of the cellular response appears to be compartmentalized in that this response was consistently observed in the renal lymph nodes but not in the spleen. Soluble protein components of the parasites responsible for these responses are likely multiple and shared by all life cycle stages.     

Lungs are a major organ site of cytomegalovirus latency and recurrence

Recurrence of infectious virus from the latent viral genomes is the initiating event in the pathogenesis of cytomegalovirus (CMV) disease during states of immunodeficiency. Interstitial pneumonia is a frequent manifestation of posttransplantation CMV disease, in particular after bone marrow transplantation and heart and lung transplantations. Recurrence can occur within the transplant derived from a latent infected donor as well as within latently infected organs of the transplant recipient. The reason for a predilection of the lungs as a site of CMV pathology is so far unknown. In a murine model of CMV latency, the lungs were identified as an authentic site of latent infection, since the viral genome remained detectable in lung tissue even after it was cleared to an undetectable level in blood and bone marrow. A comparison between the lungs and the spleen, the previously most thoroughly investigated site of murine CMV latency, revealed a 10-fold-higher burden of latent viral genome for the lungs. Most important, the organ-specific risk of in vivo recurrence was found to correlate with the organ-specific viral genomic load. This new finding thus characterizes the lungs as a high-risk organ for CMV recurrence, and this fact may explain in part why interstitial pneumonia is a frequent manifestation of recurrent CMV infection.     

Quantitative analysis of the movements of cytoplasmic granules in polarized fibroblasts

Movements of cytoplasmic organelles were analyzed in Vero fibroblasts. In the cells polarized at the edge of an experimental wound, cytoplasmic granules moved randomly (Brownian motions) and by separate jumps (saltatory movements). The displacement of granules by the Brownian motions exceeded by more than an order of magnitude that of the mitochondria similar by weight. Lipid droplets moved predominantly by saltations, whereas mitochondria and lysosomes moved much less often. In a front part of the polarized cells, the main directions of saltatory movements were from the nucleus to the leading edge of a cell and back, whereas the tangential movements (across the long axis of a cell) were less than 1%. 90% of saltatory movements occurred in the area starting 10-12 microm from the nucleus and ending 10-12 microm from the leading edge of a cell. The average rate of saltatory movements of the granules (2.38 microm/s) was identical in both directions. The average length of the track was 7.49 microm; the maximum track length reached 30 microm. An increase in the granule diameter from 0.3 to 1.4 microm resulted in a minor (statistically insignificant) decrease in the average rate of the movements. The average rate of saltatory movements of mitochondria was 1.00 microm/s, and the average track length was 6.04 microm. Therefore, mitochondria, in contrast to lipid droplets, are rigidly fixed in the cytoplasm, and the force holding mitochondria is equal to the force produced by the microtubule-associated motors. Taking into account the characteristic of the centrifugal saltations, we suggest that they are mediated by an unusual dynein.     

One fate of epimastigote forms of Trypanosoma cruzi injected in mice. An ultrastructural study

Recently, we suggested that epimastigote forms of Trypanosoma cruzi are cleared from circulation of mice by a mechanism independent of lysis and that platelets play an important role in this process. These observations prompted us to look at the fate of epimastigotes in the lung, liver, and spleen of mice injected intravenously with these parasite forms. Using transmission electron microscopy, we observed clumps of epimastigotes and platelets in direct contact with phagocytes in the lumen of capillaries. However, the platelets and parasites were probably separated before phagocytosis because only parasites were found inside the phagocytes. Indeed, most of the phagocytes, although containing epimastigotes in different stages of disintegration, contained no platelets. The removal of parasites from platelets was probably mediated by phagocytes through a mechanism similar to the removal of bacteria from the surface of erythrocytes in humans. These observations suggest that the nonvirulence of T. cruzi epimastigotes in mice is not due to lysis but probably to the inability of these parasite forms to escape destruction by the phagocytes.     

Three distinct F-actin binding sites in the Dictyostelium discoideum 34,000 dalton actin bundling protein

The Dictyostelium 34 kDa protein is an actin bundling protein composed of 295 amino acids. However, the region(s) of the molecule that bind actin filaments is (are) unknown. Studies of the cosedimentation of 125I-34 kDa protein and F-actin show that the 34 kDa protein binds to F-actin with positive cooperativity and Hill coefficients of 1.9 and 3.0, for filaments 4.9 microm and 0.6 microm, respectively. The Hill coefficient is larger for short filaments that are more efficiently bundled than long filaments, suggesting that one of the binding sites is used in interfilament contacts or contributes to filament orientation within the bundle. Three distinct actin binding sites were identified using a synthetic peptide, protein truncations, and a novel epitope library screening method. The ability to bind actin was assessed by 125I-F-actin overlays under denaturing and nondenaturing conditions, cosedimentation, viscometry, and pyrene-labeled actin disassembly. The three actin binding domains were identified as amino acids 1-123, 193-254, and 279-295. The 62 amino acid domain (193-254) can cosediment with F-actin. The estimated Kapp obtained by the disassembly of pyrene-labeled actin was 0.11 microM and 2.7 microM for the amino acids 1-123 and 279-295, respectively. These results identify three distinct regions of the 34 kDa protein that may contribute to the positive cooperative formation of F-actin bundles.     

Migration of electrons and holes in crystalline d(CGATCG)-anthracycline complexes X-irradiated at 4 K

Sialic acid acceptors of Trypanosoma cruzi are abundant mucin-like glycoproteins linked to the parasite membrane by a glycosylphosphatidyl inositol (GPI) anchor. They are heterogeneous and variable in different parasite stages. The protein portion of these mucins contains many threonine residues, and is thought to be encoded by a heterogeneous gene family. To investigate whether the high degree of heterogeneity in the mucin gene family is responsible for the diversity of mucins expressed on the parasite surface, we have studied the expression of mucin genes in several developmental stages of T. cruzi. We have found that mucins are expressed in all parasite stages. By using conserved sequences at 3' end of translated sequences of the gene family and the splice leader sequence, we have isolated 120 mucin-like cDNAs by RT-PCR from epimastigote and trypomastigote mRNAs. All transcribed genes contain conserved 5' and 3' regions, which code for the signal peptide, the sequence for GPI anchor addition, and a conserved domain rich in threonine residues. The internal portions of these genes are highly variable in size and sequence, and can be grouped in two major categories. One group contains KP(1-2)T(6-8) repeats, a motif found in mammalian mucins in the central region. This group is expressed preferentially in the trypomastigote forms ready to be released from the infected mammalian cell. The other has highly variable sequences in the central portion, and is expressed in all parasite stages. Because the number of synonymous substitutions is equivalent to the non-synonymous substitutions in the second group, they are probably evolving neutrally. On the other hand, the KP(1-2)T(6-8) containing genes have more synonymous substitutions and are most likely under a strong selective pressure. We propose that the group of KP(1-2)T(6-8) motif corresponds to the highly glycosylated mucins of the trypomastigote stages. In the other group proteolysis may remove the central domain yielding small mucins, such as the mucins found in insect derived stages of T. cruzi.     

Tenascin-C expression in human epidermal keratinocytes is regulated by inflammatory cytokines and a stress response pathway

Melanin pigments in lower vertebrates are often found in locations other than the skin, thus forming an extracutaneous pigmentary system of unknown function. The cellular and biochemical structure of this system is still poorly characterized. This paper deals with the ultrastructural and biochemical features of the melanogenic system of Xenopus laevis. Melanin containing cells were identified in the dorsal and ventral skin, and in the lung, spleen, liver and connective tissue surrounding blood vessels. The pigment cells in the skin and the lungs appeared to be typical melanocytes. The spleen contained isolated melanocyte-like cells, but most of the pigment cells present in this organ were associated with melanomacrophage centers. Conversely, the liver appeared devoid of melanocytes and only displayed melanomacrophage centers. Tyrosinase activity was found in all pigment-containing organs except the liver. All organs containing tyrosinase activity also displayed melanin formation potential from L-tyrosine. Therefore, tyrosine hydroxylase and melanin formation activities could be detected only in those organs containing typical melanocytes but not in locations such as the liver, where only melanomacrophages centers were found.     

New pathogens and mode of action of azithromycin: Toxoplasma gondii

Azithromycin can inhibit the growth of Toxoplasma gondii tachyzo?tes in vitro, but the effect is only observed with prolonged incubation with the drug, reflecting the delayed mode of action of this macrolide on the parasite. Azithromycin is probably acting by inhibition of protein synthesis but the site of action and fixation in the parasite has not been demonstrated. Azithromycin is also effective against intracystic bradyzo?tes in vitro, but long term administration of azithromycin to chronically infected mice failed to reduce the mean number of brain cysts. In models of acute toxoplasmosis, azithromycin was found to have a limited effect on brain infection, whereas parasites were cleared from blood and lungs of infected mice, resulting in a significant protection of treated mice comparatively to untreated controls. When azithromycin is combined with pyrimethamine or sulfadiazine, an additive effect is observed in vitro, and a remarkable synergistic effect is observed in vivo in the treatment of acute toxoplasmosis. Together, these results are in favor of the use of azithromycin in combined therapies for the treatment and/or prophylaxis of toxoplasmosis.     

Induced immunity to Plasmodium yoelli nigeriensis in albino mice by antigenic mice organs

Antigenic materials prepared from parasite infected and non-infected tissue (blood), organs (spleen, liver, lung) and whole mouse burnt with or without Aframomum melegmeta (Alligator Pepper) were tested whether they could elicit immune response to Plasmodium yoelli nigeriensis in albino mice. This investigation is in line with the practice of traditional medicine in the western part of Nigeria where burnt herbal preparation are introduced into patient through body cuts known as "Gbere" for protection and therapy against infection. Results from the study unexpectedly showed that immune response was elicited against malaria parasite by the uninfected antigenic material prepared from spleen and whole mouse. Aframomum melegmeta on its own lysed the red blood cells and played a doubtful role in inducing immunity.     

Effects of partial liquid ventilation on lung injury in a model of acute respiratory failure: a histologic and morphometric analysis

OBJECTIVE: To compare the histopathologic changes observed in a sheep model of oleic acid-induced acute respiratory failure during partial liquid ventilation with perflubron with gas ventilation. DESIGN: Randomized, controlled study. SETTING: Animal laboratory and pathology laboratories of a university hospital. SUBJECTS: Fourteen healthy adult sheep, weighing 64.9 +/- 6.4 kg. INTERVENTIONS: Lung injury was induced with oleic acid (0.15 mL/kg). A tracheostomy tube was inserted, along with systemic and pulmonary artery monitoring catheters. Animals were randomized to undergo either partial liquid ventilation (n = 7) or gas ventilation (n = 7). Animals underwent euthanasia at the end of the 90-min study period, after which the endotracheal tube was clamped with the lungs in expiratory hold at a positive end-expiratory pressure of 5 cm H2O. En bloc excision of the heart and lungs was performed by thoracotomy. Perfusion of the isolated lung vasculature with 2.5% paraformaldehyde and 0.25% glutaraldehyde in a 0.1-M phosphate buffer was performed. Histologic analysis followed. MEASUREMENTS AND MAIN RESULTS: Gas exchange increased markedly in the animals that underwent partial liquid ventilation compared with the gas-ventilated animals (PaO2 at 90 mins: gas ventilation-treatment group, 40 +/- 8 torr [5.3 +/- 1.1 kPa]; partial liquid ventilation-treatment group, 108 +/- 60 torr [14.4 +/- 8.0 kPa]; p = .004). Lung histologic analysis demonstrated a better overall diffuse alveolar damage score (partial liquid ventilation-treatment group, 12.4 +/- 1.4; gas ventilation-treatment group, 15.0 +/- 1.7; p = .01). In the partial liquid ventilation-treatment group, we observed an increase in mean alveolar diameter (partial liquid ventilation-treatment group, 82.4 +/- 2.9 microm; gas ventilation-treatment group, 67.7 x 3.9 microm; p = .0022) and a decrease in the number of alveoli per high-power field (partial liquid ventilation-treatment group, 25.7 +/- 0.9, gas ventilation-treatment group, 31.4 +/- 2.5; p = .0022), in septal wall thickness (partial liquid ventilation-treatment group, 6.0 +/- 0.6 microm; gas ventilation-treatment group, 8.3 +/- 1.0 microm; p = .0033), and in mean capillary diameter (partial liquid ventilation-treatment group, 13.0 +/- 0.8 microm; gas ventilation-treatment group, 19.9 +/- 1.4 microm; p = .0022). CONCLUSIONS: Partial liquid ventilation is associated with notable improvement in gas exchange and with a reduction in the histologic and morphologic changes observed in an oleic acid model of acute lung injury.     

Fc epsilon receptor-positive cells are a major source of antigen-induced interleukin-4 in spleens of mice infected with Schistosoma mansoni

When cultured in vitro with either mitogen or parasite antigens, spleen cells from mice infected with Schistosoma mansoni produce significantly higher levels of IL-4 than splenocytes from control animals. Previous studies suggested that this increase in IL-4 production occurs because of a selective expansion of T helper type 2 (Th2) cells in infected mice. However, these experiments employed unfractionated spleen populations rather than purified T lymphocytes. Here we demonstrate that T-depleted spleen cells from infected animals synthesize high levels of interleukin-4 (IL-4), but no IL-5 when stimulated with parasite antigen in vitro. Nevertheless, when purified by sorting, T cells and non-B, non-T (NBNT) populations produced similar amounts of IL-4 in response to parasite antigen. The IL-4 producing NBNT cells were found to belong to an Fc epsilon receptor (Fc epsilon R)-positive population which after sort purification produced high levels of IL-4 (between 1000 and 2000 U of per 5 x 10(3) cells). FACS analysis revealed that these Fc epsilon R+ cells make up 0.53% of splenic NBNT cells in control animals while in 8-9-week-infected animals they increase to 3.8% of that population. In contrast, in mice with 8-week unisexual worm infections these cells comprise only 1.71% of NBNT cells, indicating that eggs are a major stimulus of the response. The expansion of Fc epsilon R+ cells and their production of IL-4 could be an important factor regulating the selection and induction of different CD4+ subsets in schistosome-infected hosts.     

Feldkamp and circle-and-line cone-beam reconstruction for 3D micro-CT of vascular networks

Detailed morphometric knowledge of the microvascular network is needed for studies relating structure to haemodynamic function in organs like the lung. Clinical volumetric CT is limited to millimetre-order spatial resolution. Since evidence suggests that small arterioles (50 to 300 micrometres) dominate pulmonary haemodynamics, we built a micro-CT scanner, capable of imaging excised lungs in 3D with 100 microm resolution, for basic physiology research. The scanner incorporates a micro-focal (3 microm) x-ray source, an xyz theta stage and a CCD-coupled image intensifier detector. We imaged phantoms and contrast-enhanced rat lungs, reconstructing the data with either the Feldkamp or the circle-and-line cone-beam reconstruction algorithm. We present reconstructions using 180 views over 360 degrees for the circular trajectory, augmented with views from a linear scan for the circle-and-line algorithm. Especially for platelike features perpendicular to the rotation axis and remote from the midplane, the circle-and-line algorithm produces superior reconstructions compared with Feldkamp's algorithm. We conclude that the use of nonplanar source trajectories to perform micro-CT on contrast-enhanced, excised lungs can provide data useful for morphometric analysis of vascular trees, currently down to the 130 microm level.     

Tissue distribution of polybutylcyanoacrylate nanoparticles loaded with spin-labelled nitrosourea in Lewis lung carcinoma-bearing mice

The tissue distribution of polybutylcyanoacrylate nanoparticles (PBCN) with a diameter of 127 nm, loaded with 1-(2-chloroethyl)-3-(1-oxyl-2,2,6,6-tetramethylpiperidinyl)-1- nitrosourea (spin-labelled nitrosourea, SLCNU) is described. PBCN-suspensions were intraperitoneally (i.p.) injected into Lewis lung carcinoma bearing mice. The biodistribution of PBCN in the visceral organs, blood and tumor was studied by electron spin resonance (ESR) spectroscopy. A relatively low accumulation of nanoparticles in the liver and spleen was found. The accumulation was negligible in the i.m. implanted primary tumor. SLCNU-loaded nanoparticles were mainly found in the lungs, kidneys, and heart. The highest content of the particles studied was observed in the lungs of tumor bearing experimental animals damaged by metastases. These findings suggest that PBCN offer some opportunities in the targeting of SLCNU to lung metastases.     

Expansion of IL-3-responsive IL-4-producing non-B non-T cells correlates with anemia and IL-3 production in mice infected with blood-stage Plasmodium chabaudi malaria

A prominent switch of CD4+ T cells from Th1 to Th2 type response occurs in mice infected with the non-lethal malaria parasite Plasmodium chabaudi chabaudi AS around the time of peak parasitemia. This is reflected by a decrease in IFN-gamma- and an increase in IL-4-producing cells. The peak occurs approximately 9-10 days after infection and is accompanied by anemia. The mechanism behind the switch in Th cell response is poorly understood. We here report on the production of IL-4 from a non-T cell source during P. chabaudi infection in BALB/c mice. Flow cytometric analysis of spleen and peripheral blood leukocytes (PBL) showed a dramatic increase in the percentage of non-B non-T (NBNT) cells 9-23 days after P. chabaudi infection with peak values by day 15 (approximately 30 % of splenocytes and approximately 55 % of PBL being NBNT cells). The expansion of NBNT cells correlated closely with the appearance of a cell type secreting IL-4 and IL-6 following stimulation with IL-3 and/or cross-linking of FcgammaR. Compared to cells from uninfected animals, NBNT cells from P. chabaudi-infected mice were shown to be hyper-responsive to IL-3. The levels of the hematopoietic cytokine IL-3 were elevated in supernatants from unstimulated spleen cell cultures as well as in serum at the same time points at which NBNT cell-derived IL-4 and IL-6 were detected from spleen cultures and PBL. Thus, IL-3-responsive IL-4-producing NBNT cells may provide cytokines supporting the switch from Th1 to a Th2 response which is important for the final clearance of the parasite in P. chabaudi malaria.     

Inhibition of lymphocyte proliferation by factor(s) produced by Schistosoma mansoni

Normal spleen cells of CBA mice or Fischer rats were cultured with mitogens or allogeneic cells, together with various substances of Schistosoma mansoni origin, and thymidine uptake was measured. The proliferation (DNA synthesis) of normal lymphocytes was inhibited by the incubation product of the parasite as well as by cell-free supernatant of schistosome culture. Inhibition was obtained only when active materials were added at the beginning of the culture. Both T and B cell proliferation were inhibited. The inhibitory activity found in cell-free supernatant suggested the release by the parasite of some factor(s) interfering with lymphocyte proliferation. Moreover, serum from rats infected by S. mansoni inhibited lymphocyte proliferation also. The inhibitor(s) appeared heat resistant, dialyzable and of low molecular weight (500-1000). Incubation of normal spleen cells with S. mansoni inhibitor(s) did not enhance the release of nonspecific suppressor cell factor. The inhibition of product(s) released by the parasite could explain part of the immunosuppression status found in schistosomiasis.     

Differences in eosinophil migration into the lungs following inhaled antigen challenge of parasite-naive guinea pigs with genetically determined differences in responsiveness to a gastrointestinal nematode parasite

Parasite-naive guinea pigs with genetically determined differences in responsiveness to infection with the gastrointestinal nematode parasite Trichostrongylus colubriformis were sensitised to ovalbumin and later challenged by exposure to an ovalbumin aerosol. The resultant cellular migration into the lungs was assessed by histological examination of the lungs and enumeration of cells in bronchoalveolar lavage fluid 24 h, 72 h and 7 days later. Compared with parasite-low-responder guinea pigs, there were approximately 10 times more eosinophils in lavage fluid from parasite-high-responder animals but similar numbers of neutrophils.     

Regulated spacing of synapses and presynaptic active zones at larval neuromuscular junctions in different genotypes of the flies Drosophila and Sarcophaga

Synapses at larval neuromuscular junctions of the flies Drosophila melanogaster and Sarcophaga bullata are not distributed randomly. They have been studied in serial electron micrographs of two identified axons (axons 1 and 2) that innervate ventral longitudinal muscles 6 and 7 of the larval body wall. The following fly larvae were examined: axon 1--wild-type Sarcophaga and Drosophila and Drosophila mutants dunce(m14) and fasII(e76), a hypomorphic allele of the fasciclin II gene; and axon 2--drosophila wild-type, dunce(m14), and fasII(e76). These lines were selected to provide a wide range of nerve terminal phenotypes in which to study the distribution and spacing of synapses. Each terminal varicosity is applied closely to the underlying subsynaptic reticulum of the muscle fiber and has 15-40 synapses. Each synapse usually bears one or more active zones, characterized by dense bodies that are T-shaped in cross section; they are located at the presumed sites of transmitter release. The distribution of synapses was characterized from the center-to-center distance of each synapse to its nearest neighbor. The mean spacing between nearest-neighbor pairs ranged from 0.84 microm to 1.05 microm for axon 1, showing no significant difference regardless of genotype. The corresponding values for axon 2, 0.58 microm to 0.75 microm, were also statistically indistinguishable from one another in terminals of different genotype but differed significantly from the values for axon 1. Thus, the functional class of the axon provides a clear prediction of the spacing of its synapses, suggesting that spacing may be determined by the functional properties of transmission at the two types of terminals. Individual dense bodies were situated mostly at least 0.4 microm away from one another, suggesting that an interaction between neighboring active zones could prevent their final positions from being located more closely.     

Microgrooved subcutaneous implants in the goat

We investigated the behavior of microgrooved implants in soft tissue using polystyrene implantable disks, either smooth or microgrooved (1-10 microm) on both sides. The implants were placed subcutaneously in a goat for 1, 4, or 12 weeks. Light and transmission electron microscopy showed that fibrous capsule formation around the implants was fairly uniform. After 1 week the implants were covered with a fibrous capsule about 80 microm thick. The collagen matrix was loose, and many inflammatory cells were present. After 4 weeks the matrix was more dense and contained many newly formed blood vessels. At the implant surface a layer of inflammatory cells about 10 microm thick had accumulated. Finally, after 12 weeks the matrix had densified. One cellular layer of inflammatory cells was present at the implant surface. We carried out histomorphometric measurements of capsule thickness, inflammatory layer thickness, and the number of blood vessels. Capsule thickness appeared not to decrease with time. Further, these measurements showed that there were no differences in tissue reaction between smooth and microgrooved implants. On the basis of our observations, we suggest that 1 microm deep and 1-10 microm wide microgrooves do not influence tissue response around polystyrene implants in soft tissue.     

Experimental infection of domestic sheep with culture-derived Leishmania donovani promastigotes

Domestic sheep were intradermally inoculated with culture-derived stationary phase Leishmania donovani promastigotes. Sampling of site of inoculation, liver and spleen for 244 days showed that this parasite can stay alive in the skin for up to 28 days post-inoculation. Apart from pyrexia that was evident in all the animals for 42 days, no other symptoms of kala-azar were seen. No parasites were recovered from the visceral organs throughout the sampling period, suggesting that sheep are not susceptible to infection with L. donovani. It is therefore unlikely that sheep can be synanthropic reservoirs for this parasite.