Ultrastructural development of the sarcocyst of Sarcocystis rauschorum (Apicomplexa: Sarcocystidae) in the varying lemming Dicrostonyx richardsoni

The development of the sarcocyst of Sarcocystis rauschorum in its intermediate host was studied. Lemmings were orally administered sporocysts of S. rauschorum obtained from snowy owls (Nyctea scandiaca). Beginning at 9 days postinoculation (DPI) and at various intervals to 84 DPI, skeletal muscle tissue taken from the infected lemmings was examined by electron microscopy. At 9 DPI the sarcocysts contained few metrocytes and the cyst wall was flat. The metrocytes underwent endodyogeny, and within a few days the cyst wall of the rapidly growing sarcocyst developed numerous tubulovesicular invaginations into the electron-dense layer, and the wall had a few irregular infoldings. By 21 DPI, banana-shaped bradyzoites appeared, and by 84 DPI the mature cysts were filled with bradyzoites in groups subdivided by septa and by deep infoldings of the cyst wall. The fine structure of the wall remained simple throughout maturation, with no conspicuous invagination or protrusion. The sarcocyst produced in response to S. rauschorum is unlike those from many species of Sarcocystis, which have complex walls that change markedly as the sarcocysts mature; however, its simple appearance is similar to other species that have rodents as intermediate hosts and raptorial birds as definitive hosts.     

Diphenyleneiodonium sulfate, an NADPH oxidase inhibitor, prevents early alcohol-induced liver injury in the rat

The oxidant source in alcohol-induced liver disease remains unclear. NADPH oxidase (mainly in liver Kupffer cells and infiltrating neutrophils) could be a potential free radical source. We aimed to determine if NADPH oxidase inhibitor diphenyleneiodonium sulfate (DPI) affects nuclear factor-kappaB (NF-kappaB) activation, liver tumor necrosis factor-alpha (TNF-alpha) mRNA expression, and early alcohol-induced liver injury in rats. Male Wistar rats were fed high-fat liquid diets with or without ethanol (10-16 g. kg(-1). day(-1)) continuously for up to 4 wk, using the Tsukamoto-French intragastric enteral feeding protocol. DPI or saline vehicle was administered by subcutaneous injection for 4 wk. Mean urine ethanol concentrations were similar between the ethanol- and ethanol plus DPI-treated groups. Enteral ethanol feeding caused severe fat accumulation, mild inflammation, and necrosis in the liver (pathology score, 4.3 +/- 0.3). In contrast, DPI significantly blunted these changes (pathology score, 0.8 +/- 0.4). Enteral ethanol administration for 4 wk also significantly increased free radical adduct formation, NF-kappaB activity, and TNF-alpha expression in the liver. DPI almost completely blunted these parameters. These results indicate that DPI prevents early alcohol-induced liver injury, most likely by inhibiting free radical formation via NADPH oxidase, thereby preventing NF-kappaB activation and TNF-alpha mRNA expression in the liver.     

Low-dose meningeal worm (Parelaphostrongylus tenuis) infections in moose (Alces alces)

Parelaphostrongylosis has a rapid onset and is lethal in neonatal moose (Alces alces) when large numbers of third-stage Parelaphostrongylus tenuis larvae (L3) are given experimentally. Little is known, however, about the severity and prognosis of infections acquired naturally by accidentally ingesting terrestrial gastropods which are rarely infected and have few larvae. To investigate the relationship between infecting dose, age of moose, and severity of disease, five calves were given low doses of three to 10 L3 when five (n = 2) or 9.5 mo old (n = 3). Each of two animals initially given low doses were later challenged with a dose of 15 L3. As positive controls, two calves were given doses of 15 and 30 L3, considered to be high. All five calves given low doses showed abnormal locomotory signs at 20-28 days postinoculation (DPI) that progressively became more pronounced with hind quarter weakness and front lameness. However, after 77-130 DPI, signs diminished markedly in two of these animals and disappeared in another two. Challenge infections of 15 L3 given 199 days after initial infections had no noticeable effects although an immature worm, probably resulting from the challenge, was found in the spinal cord of one animal killed 51 days later. Two positive control animals given the high doses of 15 and 30 L3 showed moderate to severe, non-resolving, locomotory signs and had to be euthanized. Results demonstrate that single, low doses of three to 10 P. tenuis L3 cause moderate disease in moose calves but over time, some worms die and animals can recover. A degree of protection may develop against future infection.     

Experimental Sarcocystis hominis infection in cattle: lesions and ultrastructure of sarcocysts

Five calves inoculated orally with 10(5)-10(6) sporocysts of Sarcocystis hominis from human feces were necropsied 10, 18, 24, 111, and 222 days postinoculation (DPI). Calves became febrile (greater than 40-41 C) between 10 and 24 DPI and developed mild anemia (packed cell volumes were reduced by 40% of initial values) between 29 and 57 DPI but otherwise remained clinically normal. Focal hepatitis, mesenteric lymphadenitis, and myocarditis were seen in calves at 10, 18, and 24 DPI. No stages of the parasite were found at any of these times except for a few merozoites in macrophages associated with myocardial lesions in the calf necropsied 24 DPI. Mature sarcocysts at 111 and 222 DPI were up to 950 microm long and their walls were up to 6 microm thick. They were found only in skeletal muscles. One immature sarcocyst was seen in the myocardium of the calf at 222 DPI.     

Photoperiod differentially affects immune function and reproduction in collared lemmings (Dicrostonyx groenlandicus)

Many nontropical rodent species experience predictable annual variation in resource availability and environmental conditions. Individuals of many animal species engage in energetically expensive processes such as breeding during the spring and summer but bias investment toward processes that promote survival such as immune function during the winter. Generally, the suite of responses associated with the changing seasons can be induced by manipulating day length (photoperiod). Collared lemmings (Dicrostonyx groenlandicus) are arvicoline rodents that inhabit parts of northern Canada and Greenland. Despite the extreme conditions of winter in their native habitat, these lemmings routinely breed during the winter. In the laboratory, collared lemmings have divergent responses to photoperiod relative to other seasonally breeding rodents; short day lengths can stimulate, rather than inhibit, the reproductive system. Male and female collared lemmings were maintained for 11 weeks in 1 of 3 photoperiods (LD 22:2, LD 16:8, or LD 8:16) that induce markedly different phenotypes. Following photoperiod treatment, cell-mediated immune function as assessed by delayed-type hypersensitivity reactions was elevated in lemmings housed in LD 16:8 and LD 8:16 relative to LD 22:2. However, antibody production to a novel antigen was unaffected by photoperiod. Exposure to LD 8:16 induced weight gain, molt to a winter pelage, and in contrast to previous studies, regression of the male, but not the female, reproductive tract. In conclusion, these data indicate that components of immune function among collared lemmings are responsive to changes in day length.     

Rodents are not a source of endogenously-produced, fecally-transmitted Caryospora bigenetica oocysts.

Fifteen Swiss-Webster mice (Mus musculus) and eight cotton rats (Sigmodon hispidus) were inoculated orally with Caryospora bigenetica oocysts. Feces from these animals were collected from 0 to 180 days postinoculation (DPI) and examined for endogenously-produced oocysts using Nomarski microscopy. Oocysts were recovered from mouse feces at 0, 1, 2, 3, 5, 7, 8, 10, and 14 DPI, and from cotton rat feces at 1, 2, and 9 DPI. The recovered oocysts were determined to be from the original inocula due to the presence of thick walls, polar granules, and Stieda and substieda bodies. All animals exhibited clinical signs at 8 DPI. Developmental stages of C. bigenetica were identified in various tissues of seven cotton rats found dead at 9, 10, 11, 12, and 13 DPI. Caryocysts were found in muzzle, tongue, footpad, scrotum, and rectum of mice and cotton rats at 30 DPI. Fecal samples collected from mice on 0, 8, 10, 12, 14, 16, and 18 DPI, and from cotton rats on 0, 9, 11, 13, 15, and 17 DPI were injected subcutaneously into 13 mice. Of the 13 mice, a Caryospora infection was observed only in the mouse inoculated with 0 DPI mouse feces. We propose that endogenously-produced C. bigenetica oocysts are not fecally-transmitted by Swiss-Webster mice or cotton rats.     

Ingested (oral) tocilizumab inhibits EAE

BackgroundBlocking the activity of IL-6 can inhibit autoimmune diseases such as rheumatoid arthritis and Crohn’s disease.ObjectiveWe examined whether an antibody against IL-6, tocilizumab (TCZ) (Actemra®), used clinically in rheumatoid arthritis (RA) would have similar anti-inflammatory effects in EAE after oral administration.Design/methodB6 mice were immunized with MOG peptide 35–55 and gavaged with control saline or TCZ during ongoing disease. Splenocytes, CD4⁺ T cells or macrophages/monocyte lineage cells (CD11b⁺) from control fed or TCZ fed mice were adoptively transferred into active MOG peptide 35–55 immunized recipient mice during ongoing disease. Actively fed and recipient mice were examined for disease inhibition, inflammation, and cytokine responses.ResultsIngested (oral) TCZ inhibited ongoing disease and decreased inflammation. Adoptively transferred cells from TCZ fed donors protected against actively induced disease and decreased inflammation. There was a decrease in IL-6 in actively treated spleen, decrease in TNF-α, Th1-like cytokine IL-12 and increase in Th2-like cytokine IL-10 in active fed and adoptively treated recipients.ConclusionsIngested (orally administered) TCZ can inhibit disease, CNS inflammation, decrease pro-inflammatory Th1-like cytokines and increase Th2-like anti-inflammatory cytokines.     

Persistence and accumulation of (potential) single strand breaks in liver DNA of rats treated with diethylnitrosamine or dimethylnitrosamine: correlation with hepatocarcinogenicity.

Effects of diethylnitrosamine (DEN) and dimethylnitrosamine (DMN) on the sedimentation pattern of [3H]thymidine-labelled Sprague-Dawley female rat liver DNA in alkaline sucrose gradients were studied with regard to time and dose dependency. In experiments at 1--56 days after a single injection it was observed that (potential) single strand breaks induced by DEN were repaired at a low rate. At 56 days the sedimentation pattern was still grossly abnormal. Half-life values of 27 and 46 days were observed after 134 mg/kg DEN (approx. 45% of the LD50) and 13.4 mg/kg DEN, respectively. Identical experiments after DMN (10 mg/kg, corresponding to about 35% of the LD50) showed return to (almost) completely control sedimentation patterns within 56 days after injection (t 1/2 = 8 days). Experiments at 6 or 56 days after the last of a series of 5 or 10 weekly injections of DEN (13.4 mg/kg) showed that a major part of DEN-induced damage (measured as single strand breaks) is of a persistent and accumulating character. No accumulation of DMN-induced rat liver lesions was observed. It is concluded that DNA fragmentation and lack of DNA repair is not a consequence of hepatotoxicity. Since at equimolar doses DEN gives appreciably less DNA alkylation (including O6-alkylguanine) but is much more effective both as an inducer of preneoplastic liver lesions and as a hepatocarcinogen when compared with DMN, we believe that the formation of persistent (and accumulating) DNA damage after DEN administration might be relevant in the process of liver tumour formation.     

L3T4(CD4)-, Lyt-2(CD8)- and Mac-1(CD11b)-phenotypic leukocytes in murine cryptococcal meningoencephalitis

An immunohistological study of L3T4(CD4)+ and LYT-2(CD8)+ lymphocytes, Mac-1(CD11b)+ monocytes and granulocytes in experimental murine cryptococcal meningoencephalitis was conducted. To assess the concomitant inflammatory reaction in an extracerebral site, livers were examined in parallel. Mice were infected i.v. withCryptococcus neoformans, group A/D, and organs were examined immunohistologically for CD4-, CD8- and monocyteand granulocyte-specific CD11b-phenotypic leukocytes over a period of 60 days. Intracerebrally, agglomerations of cryptococci formed pseudocysts that were surrounded by CD4+ and CD8+ lymphocytes at the end of the second week post-infection, followed by the invasion of monocytes and granulocytes into the lesions. After the fourth week post-infection, most of the invaded lesions were transformed into glious scars. Meningitis was usually marked and showed a homogenous distribution of CD4-, CD8- and CD11b-phenotypic cells, with a predominance of monocytes and CD4+ lymphocytes. Inflammatory infiltrates in the liver were found already 4 days post-infection. CD4+ lymphocytes and monocytes were distributed homogenously in the infiltrates, with a lower number of CD8+ lymphocytes being located rather in the periphery of the infiltrates. Comparing leukocyte kinetics in brain and liver, an important observation was the delayed immigration of immune cells at the intracerebral cryptococcal lesions as compared with the liver, and the different migration patterns of T-lymphocyte subgroups and macrophages. These results suggest that there are differential leukocyte migration patterns in the liver and brain following disseminated cryptococcosis. The immunological aspects of the observed leukocyte kinetics are discussed.     

Common antigen of oval and biliary epithelial cells (A6) is a differentiation marker of epithelial and erythroid cell lineages in early development of the mouse

Abstract. The A6 antigen - a surface-exposed component shared by mouse oval and biliary epithelial cells - was examined during prenatal development of mouse in order to elucidate its relation to liver progenitor cells. Immunohistochemical demonstration of the antigen was performed at the light and electron microscopy level beginning from the 9.5 day of gestation (26–28 somite pairs).
Up to the 11.5 day of gestation A6 antigen is found only in the visceral endoderm of yolk sac and gut epithelium, while liver diverticulum and liver are A6-negative. In the liver epithelial lineages A6 antigen behaves as a strong and reliable marker of biliary epithelial cells where it is found beginning from their emergence on the 15th day of gestation. It was not revealed in immature hepato-cytes beginning from the 16th day of gestation. However weak expression of the antigen was observed in hepato-blasts on 12–15 days of gestation possibly reflecting their ability to differentiate along either hepatocyte or biliary epithelial cell lineages.
Surprisingly, A6 antigen turned out to be a peculiar marker of the crythroid lineage: in mouse fetuses it distinguished A6 positive liver and spleen erythroblasts from A6 negative early hemopoietic cells of yolk sac origin. Moreover in the liver, A6 antigen probably distinguishes two waves of erythropoiesis: it is found on the erythroblasts from the 11.5 day of gestation onward while first extravascular erythroblasts appear in the liver on the 10th day of gestation. Both fetal and adult erythrocytes are A6-negative.
In the process of organogenesis A6 antigen was revealed in various mouse fetal organs. Usually it was found on plasma membranes of mucosal or ductular epithelial cells. Investigation of A6 antigen's physiological function would probably explain such specific localization.     

Cytogenetics of collared lemmings (Dicrostonyx groenlandicus). II. Meiotic behavior of B chromosomes suggests a Y-chromosome origin of supernumerary chromosomes

The patterns of synapsis and chiasma formation of the B chromosomes of male collared lemmings (Dicrostonyx groenlandicus) were analyzed by light and electron microscopy and compared to expectations for various hypotheses for the intragenomic origin of supernumerary chromosomes. Pachytene analysis revealed a variety of synaptic configurations including B-chromosome univalents, bivalents and trivalents. In approximately one-half of the pachytene nuclei examined, B chromosomes were in synaptic associations with the normally unpaired portion of the Y chromosome. The B-chromosome configurations at pachynema, including those involving the Y chromosome, were maintained into diakinesis and metaphase I. The meiotic behavior of the B chromosomes was inconsistent with their derivation from centric-fusion products, isochromosome formation, small-autosome polysomy, or the X chromosome. However, the frequent synapsis and apparent recombination between B chromosomes and the Y chromosome implicate this sex chromosome as a possible source of the B chromosomes in collared lemmings.     

Administration of neutralizing antibodies to interleukin-6 (IL-6) reduces experimental autoimmune encephalomyelitis and is associated with elevated levels of IL-6 bioactivity in central nervous system and circulation.

BACKGROUND: We previously demonstrated the local production of the pleiotropic cytokine interleukin-6 (IL-6) in the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE), an animal model for the human disease multiple sclerosis. MATERIALS AND METHODS: To assess the role of IL-6 in autoimmune CNS inflammation, we administered neutralizing antibodies to IL-6 in the EAE model. Their effect was examined at the clinical and histopathological level. Levels of administered antibody and IL-6 bioactivity were followed in serum and cerebrospinal fluid (CSF). RESULTS: Systemically administered antibodies penetrated into the fluid CSF in animals in which EAE was induced. Administration of anti-IL-6 reduced the development of actively induced as well as adoptively transferred EAE and was associated with increased levels of IL-6 activity in the CSF and to a lesser extent in the serum. Anti-IL-6 was still effective when given 1 day before the onset of disease signs in adoptively transferred EAE. The disease-reducing effect of anti-IL-6 was also reflected at the pathological level by the absence of inflammatory infiltrates in the CNS. CONCLUSIONS: Our study indicates that IL-6 plays an important role in autoimmune CNS inflammation. However, due to the complex nature of the in vivo interactions of administered antibodies, the disease-reducing effect of the anti-IL-6 antibodies could be caused by neutralization of IL-6 activity or by enhancement of IL-6 activity via induction of higher IL-6 levels in the CNS.     

Diminished interleukin 6 (IL-6) production during scarless human fetal wound repair

Fetal wound healing is characterized by minimal inflammation and scarless repair. IL-6 stimulates inflammation in postnatal wound healing. We hypothesized that fetal skin has a diminished IL-6 response and that exogenous IL-6 will result in scar formation. Human adult or fetal skin was placed subcutaneously in SCID mice and incisionally wounded. Wounds were excised after 4, 12, 24 or 72 h for IL-6 mRNA quantification by RT-PCR. In other grafts, 5 microgram of IL-6 was injected at wounding and then harvested at 7 days for analysis of scar formation. IL-6 production was examined in primary cultures of human fetal or adult dermal fibroblasts incubated for 8 h with 0, 0.1, 1 or 10 ng/ml of PDGF-BB. IL-6 mRNA was detected 4 h after wounding in fetal and adult wounds, but by 12 h there was no IL-6 mRNA in the fetal wounds. Adult wounds had IL-6 mRNA persisting to 72 h. IL-6 administration to fetal wounds resulted in scar formation. Fetal fibroblasts produced less IL-6 protein and mRNA at all points examined (P<0.01 vs adult). Diminished production of inflammatory cytokines such as IL-6 may be responsible for the lack of inflammation seen during fetal wound healing. Diminished inflammation may provide a permissive environment for scarless wound healing.     

Phylogeography and mitochondrial DNA (mtDNA) diversity in North American collared lemmings (Dicrostonyx groenlandicus)

Variation in the nucleotide sequence of the mitochondrial control region (250 bp) and the cytochrome b region (870 bp) was examined in collared lemmings (Dicrostonyx groenlandicus) from 19 localities in northern Alaska and the Canadian Arctic. The division of D. groenlandicus in two phylogeographical groups with limited divergence across the Mackenzie River is consistent with the separation of this species in more than one refugial area located to the northwest of the Laurentide ice sheet during the last glaciation. Populations of D.groenlandicus from formerly glaciated areas are no less variable than those in nonglaciated areas. Instead, the low intrapopulation and intraregional diversity estimates in D. groenlandicus are probably a result of regional bottleneck events due to range contractions during Holocene warming events. These results are consistent with findings previously reported on collared lemmings (D. torquatus) from the Eurasian Arctic.     

1,3,7-trimethylxanthine (caffeine) may exacerbate acute inflammatory liver injury by weakening the physiological immunosuppressive mechanism

The genetic elimination of A2A adenosine receptors (A2AR) was shown to disengage the critical immunosuppressive mechanism and cause the dramatic exacerbation of acute inflammatory tissue damage by T cells and myeloid cells. This prompted the evaluation of the proinflammatory vs the anti-inflammatory effects of the widely consumed behavioral drug caffeine, as the psychoactive effects of caffeine are mediated largely by its antagonistic action on A2AR in the brain. Because caffeine has other biochemical targets besides A2AR, it was important to test whether the consumption of caffeine during an acute inflammation episode would lead to the exacerbation of immune-mediated tissue damage. We examined acute and chronic treatment with caffeine for its effects on acute liver inflammation. It is shown that caffeine at lower doses (10 and 20 mg/kg) strongly exacerbated acute liver damage and increased levels of proinflammatory cytokines. Because caffeine did not enhance liver damage in A2AR-deficient mice, we suggest that the potentiation of liver inflammation was mediated by interference with the A2AR-mediated tissue-protecting mechanism. In contrast, a high dose of caffeine (100 mg/kg) completely blocked both liver damage and proinflammatory cytokine responses through an A2AR-independent mechanism. Furthermore, caffeine administration exacerbated liver damage even when mice consumed caffeine chronically, although the extent of exacerbation was less than in "naive" mice that did not consume caffeine before. This study suggests an unappreciated "man-made" immunological pathogenesis whereby consumption of the food-, beverage-, and medication-derived adenosine receptor antagonists may modify an individual's inflammatory status and lead to excessive organ damage during acute inflammation.     

Estrogenic effect of propylparaben (propylhydroxybenzoate) in rainbow trout Oncorhynchus mykiss after exposure via food and water

The estrogenic effect of propylparaben was investigated in a rainbow trout Oncorhynchus mykiss test system. Propylparaben was administered orally to sexually immature rainbow trout every second day for up to 10 days in doses between 7 and 1830 mg kg(-1) 2 d(-1) and in the water at 50 and 225 microg l(-1) for 12 days. Plasma vitellogenin was measured before and during the exposures and the concentrations of propylparaben in liver and muscle were determined at the end of experiments. Increases in average plasma vitellogenin levels were seen at oral exposure to 33 mg propylparabenkg(-1) 2 d(-1); the most sensitive fish responded to 7 mg kg(-1). The ED(50) values for increase in vitellogenin synthesis were 35, 31 and 22 mg kg(-1) 2 d(-1) at day 3, 6 and 11, respectively. Exposure to 225 microg propylparabenl(-1) increased vitellogenin synthesis, but exposure to 50 microg l(-1) did not. Propylparaben showed little tendency to bioaccumulation in rainbow trout; less than 1 per thousand of the total amount of propylparaben administered orally at 1830 mg kg(-1) 2 d(-1) over the 10-d experimental period was retained in muscle and liver 24 h after the end of the experiment. Exposure to 225 microg propylparabenl(-1) for 12 d led to concentrations of 6700 and 870 microg propylparabenkg(-1) liver and muscle, respectively. Half lives for propylparaben were 8.6 h in liver and 1.5 h in muscle.     

Polynitroxyl albumin plus tempol attenuates liver injury and inflammation after hepatic ischemia and reperfusion

PNA+Tempol, albumin containing conjugated (polynitroxyl albumin; PNA) and free (4-hydroxyl-2,2,6,6-tetramethyl-piperidinyl-1-oxyl; Tempol) nitroxide may protect against injury caused by reactive oxygen species. Therefore, the actions of PNA+Tempol on liver injury and inflammation induced by hepatic ischemia and reperfusion (I/R) were examined. Rats were subjected to 1 h ischemia followed by 24 h reperfusion in the absence (I/R) or presence of PNA+Tempol (25%; 15 mL/kg, i.v.) (I/R+PNA+Tempol) or human serum albumin (23%; 13.5 mL/kg, i.v.) (I/R+HSA). Test solutions were administered prior to and for 2 h during reperfusion. Sham-operated rats underwent surgery with neither ischemia nor infusion. I/R+PNA+Tempol rats had significantly less liver injury and inflammation than I/R rats. I/R+PNA+Tempol livers exhibited focal lesions whereas I/R livers exhibited global necrosis. Likewise, plasma ALT activity was significantly lower in I/R+PNA+Tempol rats. PNA+Tempol reduced I/R-induced neutrophil accumulation and intercellular adhesion molecule-1 (ICAM-1) expression. HSA did not alter I/R-induced liver injury or inflammation. Sham-operated rats exhibited normal liver morphology and no inflammation. Attenuation of I/R liver injury by PNA+Tempol may be mediated by its effect on inflammation, the major contributor to I/R injury. Reduction of inflammation by PNA+Tempol is most likely due to the antioxidative nature of the nitroxides.     

Streptococcus equisimilis infection in striped skunks (Mephitis mephitis) in Saskatchewan

Three radio-collared striped skunks (Mephitis mephitis) found dead during a field study of winter ecology of striped skunks near Willowbrook, Saskatchewan, Canada were examined. Streptococcus equisimilis was identified as the primary agent causing necrotizing purulent pneumonia in one skunk and suppurative meningoencephalitis in another. Both Streptococcus equisimilis and Streptococcus canis were isolated from lesions of purulent myocarditis and pyothorax in the third skunk. These are apparently the first reported cases of S. equisimilis infection in striped skunks and suggest that this opportunistic pathogen may be a significant cause of mortality under some conditions.