Moderate protection is induced by a chimeric protein composed of leucine aminopeptidase and cathepsin L1 against Fasciola hepatica challenge in sheep

Leucine aminopeptidase (FhLAP) and cathepsin L1 (FhCL1) of Fasciola hepatica play a critical role in parasite feeding, migration through host tissue, and immune evasion. These antigens have been tested for immune protection as single components with variable degrees of success. The chimeric-protein approach could improve protection levels against fasciolosis. Previously, we reported the design and construction of a chimeric protein composed of antigenic sequences of FhLAP and FhCL1 of F. hepatica. The goal of the present study was to express and evaluate the immune-protective capacity of this chimeric protein (rFhLAP-CL1) in sheep. Animals were randomly allocated into five groups with five animals in each group. Groups 1, 2 and 3 were immunized twice with 100 μg, 200 μg and 400 μg of rFhLAP-CL1 emulsified with Quil A adjuvant, whereas groups 4 and 5 were the adjuvant control and infection control groups, respectively. The animals were then challenged with 200 metacercariae two weeks after the rFhLAP-CL1 booster. The fluke burden was reduced by 25.5%, 30.7% (p < 0.05) and 46.5% (p < 0.01) in sheep immunized with 100 μg, 200 μg and 400 μg of chimeric protein, respectively, in comparison to the infection control group. There was a reduction of 22.7% (p < 0.05) and 24.4% (p < 0.01) in fecal egg count in groups 2 and 3, respectively, compared to the infection control group. Sheep immunized with chimeric protein produced F. hepatica excretion-secretion product-specific total IgG antibody, which were increased after challenge. Moreover, the levels of rFhLAP-CL1-specific IgG1 and IgG2 isotypes in immunized sheep increased rapidly two weeks after the first immunization and were significantly more elevated than those of the control groups, indicating a mixed Th1/Th2 response. This is a preliminary evaluation of the chimeric protein rFhLAP-CL1 as a possible immunogen against F. hepatica infection in sheep.     

Cerebrospinal fluid and plasma concentrations of oxytocin and vasopressin during parturition and vaginocervical stimulation in the sheep

Simultaneous blood and cerebrospinal fluid (CSF) samples were taken from conscious sheep before, during and after parturition. Concentrations of plasma and CSF oxytocin were significantly elevated during contractions and particularly at birth. Mean prepartum CSF concentrations of oxytocin were around 55% of those found in plasma but postpartum they were up to 2-fold higher than those in plasma. Plasma concentrations of oxytocin were only significantly elevated, compared to prepartum levels, for 15 min postpartum whereas those in CSF were increased for the whole of the 120 min postpartum sampling period. Plasma, but not CSF, concentrations of arginine-vasopressin (AVP) were significantly raised during contractions and birth, and for 15 min postpartum. During the prepartum period CSF AVP concentrations were 67% of those found in plasma whereas at birth plasma levels were 10-fold higher than in CSF. In a separate experiment it was shown that 5 min of mechanical vaginocervical stimulation also stimulated significant increases in CSF and plasma oxytocin concentrations and in plasma vasopressin. Results support previous work suggesting an important role for central oxytocin release in the postpartum induction of maternal behavior and demonstrate that elevated concentrations of oxytocin in the CSF are present for a greater period than in blood. Elevated plasma AVP concentrations during contractions, birth or vaginocervical stimulation may be stimulated by stress associated with these stimuli.     

Postnatal differentiation and development of the rat epididymis: A stereological study

Postnatal development and differentiation of the rat epididymis was studied in the rat from 15 to 120 days of life using stereological techniques. Both the relative volume (volume density) and absolute volume of the epithelial, interstitial, and luminal compartments in the initial segment, caput, corpus, and cauda epididymides were determined. In all segments the volume density of the epithelial compartment increased between days 15 and 30 before falling to adult values at 45 days in the initial segment (0.476 ± 0.031), at 60 days in the caput (0.258 ± 0.028) and at 90 days in the corpus (0.245 ± 0.007) and cauda (0.140 ± 0.004). The relative volume of the interstitium decreased, whilst that of the lumen increased over the same period with adult values being achieved earlier in the proximal segments than in the distal segments. In contrast to volume fraction the absolute volume of all compartments in all segments increased from day 15 to day 90. Between 90 and 120 days the absolute volumes of compartments in the initial segment and caput showed little volume change. All compartments in the corpus and cauda showed significant increases in volume over the same period. A similar pattern of development was observed with respect to the surface area of both the luminal and basement membrane aspects of the epithelium; surface area per unit volume (surface density) in all segments reached adult values at approximately 60 days, whilst the increase in absolute area of the surfaces ceased at 90 days in the initial segment and caput and continued to 120 days in the corpus and cauda. The total length of the epididymal tubule showed the same pattern with no increase in length apparent in the initial segment and caput after 90 days. Length however continued to increase in the corpus and cauda between 90 and 120 days. Tubule and luminal diameter reached their definitive values in the initial segment on day 60 but continued to increase until day 90 in the distal segments. Epithelial height increased between 15 and 30 days in all segments; in the caput, epithelial height was greatest on day 30 before decreasing slightly; and in the corpus and cauda maximal epithelial height was observed on day 45 decreasing to stable values on day 60 and 90, respectively. All the data is consistent with a proximal to distal differentiation and development of the epididymis with growth continuing in all segments after the establishment of the definitive architecture. Growth of the distal segments continued after its cessation in the proximal segments. © 1994 Wiley-Liss, Inc.     

Surface coat of sheep pulmonary intravascular macrophages: Reconstitution,and implication of a glycosyl-phosphatidylinositol anchor

Background: Pulmonary intravascular macrophages (PIMs) of sheep have a globular surface coat that facilitates endocytosis of tracer particles and Escherichia coli lipopolysaccharide, and is disrupted by the heparin and Brefeldin A treatments. The present study investigated the in vivo dynamics of the coat globules following heparin-mediated removal, and the mechanism of globule organization on the plasma membrane of PIMs in vitro. Methods: Sheep were administered heparin at a dose of 50 IU/kg body weight IV, and euthanised at 30 min, 3, 6, 12, 48, and 120 hr (n = 2 for each treatment) after the treatment. Control sheep (n = 2) were injected with normal saline solution. The tissues were processed for an ultrastructural examination and acid phosphatase (ACPase) cytochemistry. Heparintreated lungs were subjected to morphometric analysis of the coat globules. Lung tissues from normal sheep (n = 2) were incubated with phosphatidylinositol-specific-phospholipase C (PIPLC; 2 IU/ml PBS) in vitro for 30 and 75 min. Results: Heparin study: The ultrastructural and morphometric data showed that the coat globules were removed at 30 min and reconstituted within 48 hr of the treatment. The PIMs showed priminent Golgi complexes associated with secretory vesicles, microtubules, and centriole between 3–12 hr of heparin treatment. Acid phosphatase cytochemistry also demonstrated secretory activity in the Golgi complexes of PIMs during the coat reconstitution. PIPLC study: The coat globules of PIMs were removed in a time-dependent mode by the PIPLC treatment without damage to other cell organelles. Conclusions: This study demonstrates a time-dependent reconstitution of the coat of PIMs in conjunction with secretory activity following heparinmediated removal, probably through sequenstration of the globules from blood. This ability is of functional significance as the coat mediates particle endocytosis by the PIMs. The results also suggest the presence of a glycosyl-phosphatidylinositol (GPI) anchor in tethering of globules on the plasma membrane of PIMs to offer a structural basis for their integrity in pulmonary vascular flow. © 1995 Wiley-Liss, Inc.     

Cerebral sodium sensors in the sodium-deplete sheep

The sodium intake of sodium deplete sheep was studied during local, push-pull perfusion of different solutions within the third cerebral ventricle. Sheep were made sodium deplete by continuous loss of parotid saliva, and were allowed access to 0.6 M NaHCO₃ solution for 2 h daily. Local perfusion within the third cerebral ventricle was performed before and during the access to sodium solution. Four perfusion sites were used: anterior dorsal and ventral, and posterior dorsal and ventral. Perfusion of 200 mM Na-csf caused a decrease in sodium intake at each perfusion site. Perfusion of ouabain, 10⁻⁶M, caused a reduction in sodium intake only during perfusions within the anterior portion of the third ventricle. The results may indicate that specific neuronal elements sensitive to changes in intracellular sodium concentrations are located around the anterior portion of the third cerebral ventricle. These neurones, however, are not exclusive sites from where sodium intake of sodium deplete sheep can be influenced.     

A model of sterile vesicoureteric reflux in the sheep

Eighteen Coopworth ewe lambs were divided into three groups based on the initial cystourethrogram and cystometry findings at 5 – 7 weeks of age: group 1, 6 lambs with spontaneous low-pressure bilateral vesicoureteric reflux (VUR) on bladder filling were used to study the natural history of reflux; group 2, 5 lambs with no VUR detected were used to establish an experimental model of bilateral VUR using an unroofing surgical procedure; group 3, 7 lambs with spontaneous VUR detected during micturition had the same surgical procedure to increase the degree of VUR. All three animal groups were followed for 4 – 10 months. Spontaneous VUR was demonstrated in 13 of 18 lambs (25/36 ureters). The presence and severity of spontaneously occurring reflux in group 1 lambs diminished with increasing age. VUR was created successfully in group 2 and increased in degree in group 3 animals. The only significant histological finding in all three animal groups with grades II and III VUR was distal renal tubular dilatation. The sheep is a useful and readily available animal for studying VUR. During 4 – 10 months of follow-up, sterile reflux without bladder outflow obstruction resulted in distal renal tubular dilatation, but no renal parenchymal damage. Received April 17, 1997; received in revised form August 5, 1997; accepted August 21, 1997     

Effect of mebendazole againstEchinococcus granulosus andTaenia hydatigena cysts in naturally infected sheep and relevance to larval tapeworm infections in man

The ability of three treatment schedules of mebendazole to kill well-established hydatid cysts was studied. Pregnant sheep, naturally infected withEchinococcus granulosus and/orTaenia hydatigena, were treated daily with mebendazole at a dose rate of 50 mg/kg body weight for either five days, one month, or three months.At autopsy, seven months after the commencement of treatment, no evidence was found that the 5-day treatment schedule had any damaging effect onE. granulosus cysts. The effects of the one month treatment were equivocal. There was evidence of a damaging effect from the 3-month treatment schedule and protoscoleces were not infective to dogs. NoT. hydatigena cysts survived the 1- and 3-month treatments, but organisms from the 5-day treatment were infective to dogs.These results forE. granulosus in sheep suggest that long-term treatment with mebendazole may be required in hydatid disease in man. The results obtained forT. hydatigena in sheep are discussed in relation to the treatment of cysticercosis fromT. solium in man. Mebendazole showed no untoward effect on the sheep or their lambs.     

Medullary control of the pontine swallowing neurones in sheep

Summary The origin of the inputs from the medullary swallowing centre (dorsal region including the nucleus of the solitary tract, or ventral region corresponding to the reticular formation surrounding the nucleus ambigous) to the pontine swallowing neurones (PSNs) was studied in sheep anaesthetized with halothane.Out of 101 PSNs located in the posterior part of the trigeminal (Vth) motor nucleus, 46 were activated by stimulating either the dorsal (21 neurones) or the ventral (25 neurones) region of the ipsilateral medullary swallowing centre, 3–4 mm rostral from the obex. Thirty-one neurones out of the 46 were identified as a motoneurones supplying swallowing muscles (mylohyoïd, anterior body of digastric and medial pterygoïd). Their average activation latency through stimulation of the dorsal medullary region was about 1 ms longer than through stimulation of the ventral region (3.63 ms±0.81 versus 2.72 ms±0.32).To determine the origin of the medullary input to the PSNs, we tried to activate the medullary swallowing neurones (MSNs) antidromically through stimulating the posterior part of the Vth motor nucleus, which contains the swallowing motoneurones. Seventy-three MSNs were tested (25 located in the dorsal and 48 in the ventral region). None of the dorsal neurones tested could be antidromically activated by pontine stimulation: 15 ventral neurones showed a clear antidromic response (collision test) with an average latency of 2.5 ms±0.73. These neurones, which send their axons into the pons, were all located in the reticular formation, above the nucleus ambiguus, 3–4 mm rostral from the obex.These results suggest that MSNs in the ventral reticular formation connect the medullary swallowing centre to the Vth motor nucleus. They also suggest that during swallowing, inputs originating from the dorsal region of the medullary centre (interneurones programming the motor sequence) are relayed in the ventral region (reticular formation adjacent to the nucleus ambiguus) before reaching the PSNs.This work was supported, in part, by grants from CNRS (LA 205), INRA and M.R.I. (82 E 0685)     

Frequencies of background immunoglobulin-secreting cells in mice as a function if organ, age, and immune status

The influence of hereditary absence of thymus and spleen upon the numbers, organ, and class distribution of background immunoglobulin Ig-secreting cells was studied in mice by means of the protein-A plaque assay. In young adult BALB/c mice the spleen contained the largest number of Ig-secreting cells (about 0.5% ). The absolute number of Ig-secreting cells in the spleen was larger than the estimate for all lymph nodes together. Between 8 and 40 weeks of age, the number of Ig-secreting cells in spleen and lymph nodes increased by a factor of 3, maximally. In the same period, the number of Ig-secreting cells in the bone marrow, however, increased by a factor of 20, so that it became the major site of Ig synthesis. Hereditary absence of the spleen did hardly or not at all affect the number of Ig-secreting cells in the other lymphoid organs. However, the athymic state did affect the organ distribution. The most consistent finding was the decreased number of Ig-secreting cells in the Peyer's patches.The class distribution of Ig-secreting cells was found to be independent of the presence of the spleen, but did depend on the presence of the thymus. Athymic mice had a higher percentage of IgM-secreting cells and a lower percentage of IgA-secreting cells. The percentage of IgG₁- and IgG₂-secreting cells did not differ clearly between normal and athymic mice. Percent-wise, most IgM-secreting cells occurred in the spleen, whereas most IgG₁-, IgG₂-, and IgA-secreting cells occurred in the bone marrow, lymph nodes, and Peyer's patches.The specificity repertoire of the background Ig-secreting cells was tested by determining the frequencies of IgM-producing cells with specificity for a panel of six different antigens. These frequencies ranged from 1 in 85 for nitroiodophenyl(NIP)-conjugated sheep erythrocytes (SRBC) till 1 in 1500 for unconjugated SRBC and were found to be the same for the spleen of germ-free and specific pathogen-free (SPF) C3H mice, and for spleen, bone marrow, and thymus of SPF C3H mice.     

The instability of membrane markers expressed by human monocytes and macrophages in culture

Surface markers were tested on freshly isolated human monocytes and following their in vitro maturation to macrophages. The markers tested were HLA-DR antigens, receptors for the Fc of IgG and complement as well as membrane markers defined by monoclonal antibodies. The results revealed a dynamic expression of some of the markers on monocytes which was influenced by several variables. The expression of the markers was modulated by the presence of different sera, by treatment with lymphokines and interferon and following the in vitro maturation of monocytes to macrophages. The most unstable marker was found to be the HLA-DR, which was modulated by all these variables. The 63D3 was affected by different sera and culture supernatant, as well as following the maturation of monocytes to macrophages, but not by lymphokines and interferon. One of the markers, the Mac 120, was found to be relatively stable and did not change significantly following the maturation of monocytes to macrophages. The Fc and complement receptors were also stable in their expression under these conditions, but were probably partially blocked in the presence of human serum. These results indicated that at least some of the heterogeneity related to the monocyte population was probably not due to the occurrence of stable subsets of cells, but rather to reversible changes in marker expression.