Mona Aleksandersen

The intestinal habitat for organized lymphoid tissues in ruminants; comparative aspects of structure, function and development.

Unlike the Peyers patches of rats and mice, which are considered to be secondary lymphoid organs, the ileal Peyers patch of sheep is thought to be responsible for the primary generation of B cells, like the bursa of Fabricius of birds. The ileal Peyers patch of sheep shows prenatal maturation, antigen-independent lymphopoiesis, a rate of lymphocyte production larger than that of the thymus, and involution at a young age. Follicles contain few T cells and have an IgM+, relatively immature B lymphocyte population, as judged by B-cell differentiation markers. The follicle-associated epithelium of the ileal Peyers patch is of a special type that sheds carbonic anhydrase-rich, 50-nanometer membrane-bounded particles (carbonic anhydrase-reactive particles; CAP) into the intercellular spaces. The CAP filter into the follicle centre and are taken up by lymphocytes. They represent the epithelial (bursa-like) element in an otherwise mesenchymal stroma of reticular cells embedding the follicle lymphocytes. Transepithelial transport of macromolecules, with the formation of multivesicular body-like cytoplasmic vacuoles, appears to be the basis for CAP formation. The jejunal Peyers patches are devoid of CAP, persist in the adult animal, contain M cells with clusters of B cells in the follicle-associated epithelium, and have many CD4+ lymphocytes in the follicles and in the interfollicular areas. Aggregates of lymphoid follicles in the large intestine resemble the jejunal Peyers patches with respect to their lymphocyte population and the ileal Peyers patch with respect to their follicle-associated epithelium.

Morphological changes in the testis after long-term valproate treatment in male Wistar rats

Uncertainty exists about the effect of antiepileptic drugs on gonadal function. In females, long-term valproate treatment has been shown to induce endocrine disturbances and an increased number of ovarian cysts. The aim of the present study was to investigate whether valproate can also induce morphological changes in the testis of male animals. In addition, possible morphological changes in the liver, heart, lungs, lymphatic nodes, pancreas, kidney or brain were studied. The carcinogenic implications were evaluated by the measurement of p53. Male Wistar rats were fed perorally with valproate mixture 200 mg kg(-1)(n= 15) or 400 mg kg(-1)(n= 20), or control solution (n= 15) twice daily for 90 days. Serum concentrations measured 4-6 hours after the last dose were 105 and 404 micromol l(-1)in low- and high-dose valproate treated animals respectively. There was a highly significant, 51% decrease (P< 0.001) in testicular weight in the high-dose treated valproate rats with no changes in the other groups. There was widespread testicular atrophy with histologically verified spermatogenic arrest in 15/20 of the high-dose valproate treated animals. No changes in the testis were seen in the low-dose valproate treated rats, nor in the control rats. There were no morphological changes in the other investigated organs. None of the groups showed over-expression of p53. In conclusion, a dose-dependent effect of chronic valproate treatment was found on testicular morphology in rats. Caution must be taken before these results can be applied to humans.

Distribution of PCB 118 and PCB 153 and hydroxylated PCB metabolites (OH-CBs) in maternal, fetal and lamb tissues of sheep exposed during gestation and lactation.

The main objective of the present study was to gain knowledge about the disposition of the PCB congeners 118 and 153 in a sheep model where ewes were given oral doses of PCB in two consecutive gestations. The study aimed to investigate how maternal exposure with PCBs affected tissue concentrations in lambs and fetuses and to carry out time trend studies in ewes between gestations, as well as to increase the knowledge on metabolic processes by studying OH-metabolites. The results showed that the fetuses accumulated PCBs in levels and composition comparable to the ewes. The mean fetus/mother ratios of adipose tissue levels ranged from 0.71 to 0.82 for both PCB 118 and PCB 153 in the two exposure groups. Furthermore, the fetal brain/fat ratios ranged from 0.15 to 0.20 for both congeners. Body burden calculations (BB) showed that 16% and 43% of the total PCB 118 and PCB 153, respectively, still remained in ewes, lambs and fetuses after exposure during two gestations. The different accumulation of the two congeners is assumed to be a result of differences in metabolism. The present study demonstrate that high levels of PCB 118 and PCB 153 cross the placenta and accumulate in the fetal adipose tissue and brain thus exposing the fetus during vulnerable periods of extensive development.

Long-term levetiracetam treatment affects reproductive endocrine function in female Wistar rats

PURPOSE Several antiepileptic drugs (AEDs) induce changes in endocrine function in women with epilepsy. Levetiracetam (LEV) is one of the newer AEDs, and to date no endocrine side-effects have been reported in humans. However, a recent study on ovarian follicular cells from prepubertal pigs showed that LEV affected basal steroid hormone secretion. The aim of the present study was to investigate possible effects of the drug on endocrine function and ovarian morphology in non-epileptic rats. METHODS Thirty female Wistar rats were fed per-orally with either 50mg/kg LEV (n=15) or 150 mg/kg LEV (n=15) twice daily for 90-95 days. Twenty rats received a control solution. The rats were killed in the dioestrus phase of the oestrous cycle. Serum concentrations of testosterone, 17beta-oestradiol, progesterone, follicle stimulating hormone (FSH), luteinizing hormone (LH) and LEV were measured, and the ovaries examined histologically. RESULTS Mean ovarian weight showed a significant, dose-dependent increase after LEV treatment. Mean numbers of ovarian follicular cysts were not changed, but the numbers of corpora lutea and secondary follicles were significantly higher in the treated animals. Serum testosterone was significantly increased in treated animals (0.50 nmol/l versus 0.16 nmol/l in controls, p<0.05), while oestradiol was reduced (67.4 compared to 257.5 pmol/l in controls, p<0.05). The low-dose group had significantly lower serum progesterone concentrations than the control group (56.8 nmol/l versus 34.7 nmol/l, respectively, p<0.05). FSH was reduced in the treated animals (3.3 ng/ml versus 5.5 ng/ml, p<0.05) while LH was unaffected. CONCLUSION Our findings indicate a possible effect of LEV on the hypothalamic-pituitary-gonadal (HPG) axis and ovarian morphology in non-epileptic rats. The effects differ from those previously described for other AEDs. Caution must be taken before these results can be applied to humans.

Effects on bone tissue in ewes (Ovies aries) and their foetuses exposed to PCB 118 and PCB 153

The aim of the present study was to investigate whether low levels of mono-ortho PCB 118 and di-ortho PCB 153, affect bone composition and strength in ewes (Dala breed) and their foetuses following exposure starting at conception and ending a week before expected delivery. In male foetuses, trabecular bone mineral content at the metaphysis was almost 30% lower in the PCB 118 (49 microg/kg body wt/day) group compared to the control group (corn oil) (ANCOVA, P<0.05). In female foetuses of the PCB 153 (98 microg/kg body wt/day) group trabecular cross-sectional area at the metaphysis was 19% smaller than in the controls (ANCOVA, P<0.05). At the diaphysis a smaller marrow cavity area (up to 24% reduction) was observed in female and male foetuses exposed to PCB 153 compared with controls (ANCOVA, P<0.05). There were also significant differences at the mid diaphyseal measure point between the PCB 153 and the control group females (ANCOVA, P<0.05). Cortical and total bone mineral density, cortical thickness were significantly higher, endosteal circumference shorter and marrow cavity significantly smaller in the PCB 153 group (ANCOVA, P<0.05). In conclusion there were gender dependent effects on bone tissue and cortical bone was more affected than trabecular bone.

Phenotypic characterisation of intestinal dendritic cells in sheep

The present study was undertaken to identify dendritic cells (DCs) in the ileum and rectum of lambs and adult sheep. The distribution of these cells in four different intestinal compartments, i.e. lamina propria, lymphoid follicles, domes and interfollicular areas was assessed, and the presence of these cells in lambs and adult sheep was compared. Specimens were examined by using a number of potential DC markers (CD11c, CD205, MHC class II (MHCII), CD1b and CD209) in immunohistochemical and multicolour immunofluorescent procedures. The ovine ileal and rectal mucosa contain many CD11c+/CD205+ cells with a dendritic morphology, and the majority of these cells co-expressed MHCII. These double-positive cells were also labelled with the CD209 antibody in the lamina propria and interfollicular regions. Only very few cells expressed CD1b. In conclusion, a major DC population in ileum and rectum of sheep co-expressed the CD11c, CD205 and MHCII molecules. The CD209 antibody appeared to be a novel marker for a subpopulation of ovine intestinal DCs.

A morphological and molecular study of Anaplasma phagocytophilum transmission events at the time of Ixodes ricinus tick bite

BackgroundAnaplasma phagocytophilum is the causative agent of human granulocytic anaplasmosis (HGA) in humans and tick-borne fever (TBF) in ruminants. The bacterium invades and replicates in phagocytes, especially in polymorphonuclear granulocytes.MethodsIn the present study, skin biopsies and ticks (Ixodes ricinus) were collected from tick feeding lesions on 38 grazing lambs between two and three weeks after access to pastures. The histopathological changes associated with tick bites and A. phagocytophilum infection, were described. In addition the skin biopsies were examined by immunohistochemistry. Furthermore, samples from blood, skin biopsies and ticks were examined by serology, PCR amplification of msp2 (p44), genotyping of rrs (16S rRNA) variants, and compared with the results obtained from histological and immunohistochemical investigations.ResultsTick bites were associated with chronic and hyperplastic inflammatory skin lesions in this study. A. phagocytophilum present in skin lesions were mainly associated with neutrophils and macrophages. Bacteria were occasionally observed in the Tunica media and Tunica adventitia of small vessels, but were rarely found in association with endothelial cells. PCR and genotyping of organisms present in blood, ticks and skin biopsies suggested a haematogenous and a local spread of organisms at the tick attachment sites.ConclusionsThe present study describes different aspects of A. phagocytophilum infection at the site of tick bite, and indicates that A. phagocytophilum rarely associates with endothelium during the early pathogenesis of infection.

In utero and lactational exposure to PCB 118 and PCB 153 alter ovarian follicular dynamics and GnRH-induced luteinizing hormone secretion in female lambs

The effects of in utero and lactational exposure to two structurally different polychlorinated biphenyl (PCB) congeners on follicular dynamics and the pituitary‐gonadal axis in female lambs were investigated. Pregnant ewes received corn oil, PCB 118, or PCB 153, and offspring was maintained until 60 days postpartum. Ovarian follicles were quantified using stereology. Plasma luteinizing hormone (LH) and follicle‐stimulating hormone (FSH) were measured using radioimmunoassay before and after administration of a gonadotropin releasing hormone (GnRH) analog. PCB 118 exposure increased numbers of transitional, secondary, and the sum of secondary, early antral, and antral (Σsecondary‐antral) follicles, PCB 153 exposure only increased the number of primary follicles. GnRH‐induced LH levels were significantly elevated in the PCB 153 exposure group. We conclude that PCB 153 and PCB 118 alter follicular dynamics in lambs and modulate the responsiveness of the pituitary gland to GnRH.

Natural mixtures of persistent organic pollutants (POPs) suppress ovarian follicle development, liver vitellogenin immunostaining and hepatocyte proliferation in female zebrafish (Danio rerio)

Persistent organic pollutants such as polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) and dichlorodiphenyltrichloroethane (DDT) are present in high concentrations in livers of burbot (Lota lota) in Lake Mjøsa, Norway. In order to assess effects of such pollutants on fish gonadal morphology, female zebrafish were exposed in two generations by food to mixtures of pollutants extracted from livers of burbot from Lake Mjøsa (high and low dose) and Lake Losna, which represents background pollution, and compared to a control group. Ovarian follicle counts detected a significant decrease in late vitellogenic follicle stages in fish exposed to the Losna and the high concentrations of Mjøsa mixtures in fish from the first generation. In addition, proliferation of granulosa cells, visualized by immunohistochemistry against proliferating cell nuclear antigen (PCNA), was decreased in all exposure groups in either early or late vitellogenic follicle stages compared to control. This was accompanied by increased apoptosis of granulosa cells. There was a decrease in proliferation of liver hepatocytes with exposure to both Mjøsa mixtures. In addition, immunopositivity for vitellogenin in the liver was significantly lower in the Mjøsa high group than in the control group. When analysing effects of parental exposure, fish with parents exposed to Mjøsa high mixture had significantly higher numbers of perinucleolar follicles than fish with control parents. We conclude that long-term exposure of a real-life mixture of pollutants containing high- and background levels of chemicals supress ovarian follicle development, liver vitellogenin immunostaining intensity and hepatocyte proliferation in the zebrafish model.

Lymphocyte Depletion in Ileal Peyer’s Patch Follicles in Lambs Infected with Eimeria ovinoidalis

ABSTRACT A total of 14 lambs were experimentally infected with Eimeria ovinoidalis in two separate experiments in two consecutive years. Nine lambs served as uninoculated controls. Material was collected from the ileum 2 weeks after infection in eight lambs and 3 weeks after infection in six lambs. Lambs examined 2 weeks after infection had normal follicles. After three weeks, the follicle-associated epithelium covering the lymphoid follicles of the ileal Peyer’s patches showed fusions with adjacent absorptive epithelium, focal hyperplasia, and occasionally necrosis. Macrogametes, microgamonts, and oocysts were often found in the follicle-associated epithelium and the dome region. Various degrees of lymphocyte depletion were present in the ileal lymphoid follicles in all six infected lambs 3 weeks after infection, and four lambs had decreased follicle size. Reduced staining for leukocyte common antigen (CD45), B-cell markers, and the proliferation marker Ki-67 was present in these lambs. Application of the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling method for apoptotic cells revealed decreased staining in the ileal lymphoid follicles 3 weeks after infection. A marker of follicular dendritic cells, 5′- nucleotidase, showed increased reactivity, probably due to condensation of reticular cells following loss of follicle lymphocytes. Reduced staining for carbonic anhydrase in the follicle-associated epithelium and the domes was present in all six lambs examined 3 weeks after infection, indicating decreased production of carbonic anhydrase-reactive 50-nm particles and a decreased lymphoproliferative stimulus. In conclusion, the present study shows that severe E. ovinoidalis infection in lambs causes lesions of the follicle-associated epithelium and may result in lymphocyte depletion and atrophy of the ileal Peyer’s patch follicles.