Ontogeny of Hepatic Transporters and Drug-Metabolizing Enzymes in Humans and in Nonclinical Species.

The liver represents a major eliminating and detoxifying organ, determining exposure to endogenous compounds, drugs, and other xenobiotics. Drug transporters (DTs) and drug-metabolizing enzymes (DMEs) are key determinants of disposition, efficacy, and toxicity of drugs. Changes in their mRNA and protein expression levels and associated functional activity between the perinatal period until adulthood impact drug disposition. However, high-resolution ontogeny profiles for hepatic DTs and DMEs in nonclinical species and humans are lacking. Meanwhile, increasing use of physiologically based pharmacokinetic (PBPK) models necessitates availability of underlying ontogeny profiles to reliably predict drug exposure in children. In addition, understanding of species similarities and differences in DT/DME ontogeny is crucial for selecting the most appropriate animal species when studying the impact of development on pharmacokinetics. Cross-species ontogeny mapping is also required for adequate translation of drug disposition data in developing nonclinical species to humans. This review presents a quantitative cross-species compilation of the ontogeny of DTs and DMEs relevant to hepatic drug disposition. A comprehensive literature search was conducted on PubMed Central: Tables and graphs (often after digitization) in original manuscripts were used to extract ontogeny data. Data from independent studies were standardized and normalized before being compiled in graphs and tables for further interpretation. New insights gained from these high-resolution ontogeny profiles will be indispensable to understand cross-species differences in maturation of hepatic DTs and DMEs. Integration of these ontogeny data into PBPK models will support improved predictions of pediatric hepatic drug disposition processes. SIGNIFICANCE STATEMENT: Hepatic drug transporters (DTs) and drug-metabolizing enzymes (DMEs) play pivotal roles in hepatic drug disposition. Developmental changes in expression levels and activities of these proteins drive age-dependent pharmacokinetics. This review compiles the currently available ontogeny profiles of DTs and DMEs expressed in livers of humans and nonclinical species, enabling robust interpretation of age-related changes in drug disposition and ultimately optimization of pediatric drug therapy.

Campylorrhinus lateralis, Bilateral microphthalmia and odontoma temporalis in an Oldenburg Foal.

An Oldenburg colt with wry nose was autopsied after having lived for only 30 min. It presented cyanotic oral mucosae, underdeveloped eyes and a right-sided temporal osseous mass. The applicable nomenclature for the defects is discussed, and the potential etiopathogenesis is explored by describing the normal embryonic development of the affected body parts.

Nutritional interventions to prevent and rear low-birthweight piglets.

Selection for hyperprolific sows, as a means of increasing litter size and profit, has resulted in an increased number of low-birthweight (LBW) piglets. These LBW piglets might suffer from increased morbidity and mortality during the early neonatal period. In addition, they show reduced growth performance, meat and carcass quality, which leads to an important economic loss for the farmer in the post-natal period. Therefore, nutritional interventions can be undertaken to prevent and rear LBW piglets. In the first part of this review, the preventive strategies at the sow level will be discussed. Approaches in preventing LBW piglets are to optimize the intrauterine environment via supplementing the sow during gestation. In the second part of this review, the interventions at the piglet level will be described. To increase the survival and growth rates of LBW piglets, one must focus on ensuring adequate colostrum and milk intake. Interventions include supplementing piglets, split nursing, split weaning and cross-fostering. Additional interventions increasing the probability of optimal post-natal food intake will be discussed.

Hair or salivary cortisol analysis to identify chronic stress in piglets?

Hair cortisol might better represent chronic stress than salivary cortisol in piglets. To test this hypothesis, 24 female, 7-day old piglets were allocated to two groups and artificially reared. The piglets in the stressed group were exposed to overcrowding (0.10m2/piglet) and frequent mixing with unfamiliar piglets until the age of 28 days. The control group remained in an unchanging group at a density of 0.29m2/piglet. After 3 weeks, stressed animals had gained significantly less weight (median, here and throughout, 7.58kg) than the control animals (6.43kg; P=0.021). Additionally, hair from the stressed group contained significantly higher cortisol concentrations (87.29 vs. 75.60pg/mg hair; P=0.005), whereas salivary cortisol concentrations did not significantly differ between groups (0.30 vs. 0.25 µg/dL saliva; P=0.447). Weight gain and hair cortisol concentrations were significantly correlated (P=0.036, r=-0.430), but neither of these parameters were correlated with salivary cortisol concentrations (P=0.929, r=0.019 and P=0.904, r=0.026, respectively).

Anatomy of the lower respiratory tract in domestic birds, with emphasis on respiration.

This manuscript describes the anatomy of the lower respiratory tract in domestic bird species including the chicken and pigeon. The here described anatomical structures play a major role avian respiration, which is fundamentally different from respiration in mammals. During inspiration and expiration, a continuous caudocranial airflow is present within the tertiary bronchi of the Paleopulmo, while the Neopulmo, which is only present in phylogenetically recent species, is characterized by tidal respiration. Various anatomical structures and aerodynamic mechanisms have been described in an attempt to explain the proposed mechanism of respiration. The air sac system that is essential for avian respiration usually comprises an unpaired clavicular air sac and paired cervical, cranial and caudal thoracic, and abdominal air sacs. The latter are by far the larger and are interwoven with the abdominal organs.

Anatomy of the upper respiratory tract in domestic birds, with emphasis on vocalization.

This work reviews the anatomy of the upper respiratory tract in domestic birds including the chicken and pigeon. Non-exhaustive additional information on other bird species, illustrating the extraordinary diversity in the biological class Aves, can be found in several footnotes. The described anatomical structures are functionally considered in view of avian sound production. In particular, the Syrinx is invaluable. Its most important structures are the Labia and the lateral and medial tympaniform membranes in non-songbirds and songbirds, respectively. These structures produce sound by vibrating during expiration and eventually inspiration.

Effect of artificial rearing of piglets on the volume densities of M cells in the tonsils of the soft palate and ileal Peyer's patches.

The high prolificacy of modern hybrid sows has increased the mean litter size during the last decades. However, rearing large litters is challenging and has increased the use of alternative management strategies such as euthanasia of weak piglets, cross-fostering, supplementing piglets with milk, split-nursing and split-weaning. The latter includes artificial rearing on brooders where piglets have ad libitum access to milk replacer. The effect of this artificial rearing on the immune system of the piglet is the subject of various studies. The present study focused on the M cells in the tonsil of the soft palate and in the ileal Peyer's patch (iPP). These epithelial cells are specialized in antigen sampling and play a pivotal role in the induction of adaptive immune responses. The volume densities of the M cells were assessed by stereological analysis of tissue samples from piglets of 0, 3, 8 and 19days of age. During the first three days, piglets suckled the sow, permitting them to ingest colostrum. At the third day, the piglets were either allowed to continue to suckle the sow or were transferred to brooders. The six experimental groups, each containing six piglets, thus consisted of newborn piglets, 3-day-old sow-suckled piglets, and conventionally and artificially reared piglets of 8 and 19days of age. To identify M cells, tissue samples were immersed in 4% phosphate-buffered paraformaldehyde and paraffin sections were immunohistochemically stained against cytokeratin 18. The volume densities of M cells in both the crypt epithelium of the tonsils of the soft palate and the follicle-associated epithelium of the iPPs did not show any difference between the various age groups of conventionally reared piglets. However, values were twice as high in the iPPs compared to the tonsils of the soft palate. In contrast, a decrease in volume densities of M cells was observed in the iPPs of piglets after they had been transferred to commercial brooders (P=0.05), resulting in significantly lower values (P=0.04) in comparison with the age-matched sow-suckled groups. However, this observation did not translate to values of the tonsils where M cell volume densities remained the same in all age and rearing groups. Based on these results, it appears that antigen sampling is possible from birth onwards and is more advanced in the small intestine than in the oropharynx, but possibly lags behind in artificially reared piglets.

Cell-specific localisation of apoptosis in the bovine ovary at different stages of the oestrous cycle.

Apoptosis was localized in all ovarian cell types of 23 cows in various stages of the oestrous cycle, using the detection of active caspase-3, in situ end labelling (TUNEL) and DNA fluorescent staining (DAPI). Very few apoptotic cells were found in primordial, primary, secondary and vital tertiary follicles. In contrast, apoptosis in atretic tertiary follicles was much more frequent, and high apoptotic scores were recorded when using the TUNEL technique and lower scores with the caspase-3 assay. Cystic atretic follicles showed in general a higher apoptotic score than obliterative atretic follicles, with intermediate to high scores in granulosa cells and lower scores in theca cells. In corpora lutea, large and small lutein cells had intermediate to high scores using the caspase-3 assay, and intermediate to low scores using the TUNEL assay. Irrespective of the detection method, the scores were higher in lutein cells than in the capsular stroma cells. In all ovarian structures examined, variations in apoptotic scores were seen in the different cycle stages, suggesting a cycle-dependent influence on apoptosis, although correlations with plasma progesterone concentrations were low.

Biological and chemical approaches for the detection and identification of illegal estrogens in water-based solutions.

The continuous introduction of new products used as growth promoters in animal husbandry, for sports doping and as products for body-building requires residue laboratories to initiate research on developing a strategy for the identification of 'unknown' components. In this study, a strategy is presented for elucidating the identity, the structure and the possible effects of illegal estrogenic compounds in an unidentified water-based solution. To obtain complete information on the composition and activity of the unidentified product, a multidisciplinary approach was needed. A case-study is described with a 'solution X' found during a raid. First, in vivo techniques (animal trials with mice, anatomical and histological research) were combined with in vitro techniques (the yeast estrogenic screen (YES)). In a later stage of the investigation, HPLC-fractionation, liquid chromatography-multiple mass spectrometry (LC-MSn) and gas chromatography-multiple mass spectrometry (GC-MSn) were used. Finally, the identity of 'solution X' was confirmed in a very low concentration range (10 ng/L estrone and 400 ng/l ethinyloestradiol).

[Negligible amounts of cholesterol-raising diterpenes in coffee made with coffee pads in comparison with unfiltered coffee]. / Verwaarloosbare hoeveelheden cholesterolverhogende diterpenen in koffie gezet met de koffiepadzetmethode bij vergelijking met ongefilterde kookkoffie.

OBJECTIVE: To determine the amounts of the serum-cholesterol raising diterpenes cafestol and kahweol in coffee made with coffee pads and the Senseo coffee machine as opposed to filtered and unfiltered coffee. DESIGN: Observational. METHOD: In five cities in the Netherlands coffee was purchased in three major supermarkets resulting in a total of 30 samples of coffee pads. The levels of cafestol and kahweol were determined by gas chromatography. As controls, the diterpene levels in filtered and unfiltered coffee were also measured. RESULTS: Coffee prepared using coffee pads contained on average 0.76 mg/l cafestol (95% CI: 0.69-0.82) and 0.85 mg/l kahweol (95% CI: 0.77-0.94). Filtered coffee contained 0.76 mg/l cafestol (95% CI: 0.63-0.88) and 0.81 mg/l kahweol (95% CI: 0.63-0.99). Unfiltered coffee contained 72.5 mg/l cafestol (95% CI: 48.5-96.4) and 71.5 mg/l kahweol (95% CI: 45.0-98.1). CONCLUSION: Coffee prepared using coffee pads and the Senseo coffee machine contained minute levels of diterpenes comparable to those of filtered coffee. Its effect on serum-cholesterol levels is therefore likely to be negligible.

Left Retrocaval Ureter around the Ipsilateral Limb of a Double Caudal Vena Cava in a Cat.

Necropsy examination of an adult neutered male cat, which was humanely destroyed as it had feline immunodeficiency virus (FIV) infection, demonstrated the presence of a left retrocaval ureter that was entrapped around the left limb of a double caudal vena cava. These associated anomalies originate from the complex embryofetal development of the caudal vena cava. Since no clinical signs had been reported and no gross lesions related to this anomaly were observed, this manifestation should be simply considered as an anatomical variation.

The ligaments of the canine hip joint revisited.

Numerous conventional anatomical textbooks describe the canine hip joint, but many contradictions, in particular regarding the ligament of the femoral head, are present. This paper presents a brief overview of the different literature descriptions. These are compared with own observations that have resulted in a revised description of the anatomy of the ligament of the femoral head in the dog. To this purpose, the hip joints of 41 dogs, euthanized for reasons not related to this study and devoid of lesions related to hip joint pathology, were examined. It was observed that the ligament of the femoral head is not a single structure that attaches only to the acetabular fossa, as generally accepted, but it also connects to the transverse acetabular ligament and is complemented by a strong accessory ligament that courses in caudal direction to attach in the elongation of the acetabular notch that extends on the cranioventral surface of the body of the ischium. The description of this accessory ligament in conventional anatomical handbooks is incomplete. This description of the accessory ligament of the femoral head could support the research unravelling the etiopathogenesis of hip instability.

Apoptosis in the canine endometrium during the estrous cycle.

Apoptotic cell death in the endometria of 58 female dogs in different stages of the estrous cycle was assessed (in formalin-fixed, paraffin-embedded sections) with both the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay and immunohistochemical detection of caspase-3 activity. For both techniques, the apoptotic index was determined in the surface epithelium, stroma, crypts, and basal glands by counting the percentage of stained cells in a total of 500 cells in each category. In the surface epithelium and stroma, TUNEL- and caspase-3-positive cells were rare (apoptotic index<1) throughout the estrous cycle. However, caspase-3 detection showed a significant increase in the apoptotic index in the stroma during anestrus as well as an increase in the index in both the stroma and surface epithelium in late metestrus. The apoptotic index increased during late metestrus and anestrus in the crypts and basal glands; in the crypts, this increase was significant only when caspase-3 detection was used, whereas in basal glands, significant differences were found for both techniques. In conclusion, apoptosis was present in canine endometrial cells during the estrous cycle, but caspase-3 detection showed more significant differences than the TUNEL assay. Furthermore, a high apoptotic index (suggestive of endometrial desquamation) was not detected in the surface epithelium and there was no significant correlation between the apoptotic index in any cell group and serum progesterone concentrations.

Proliferation patterns in the canine endometrium during the estrous cycle.

The proliferative activity in the endometrium of 58 bitches in different stages of the estrous cycle was assessed by immunohistochemical detection of the Ki-67 proliferation associated nuclear antigen and by counting mitotic figures. The Ki-67 labelling index and the mitotic index were determined in the surface epithelium, the stroma, the crypts and the basal glands by calculating the percentage of Ki-67 positive cells and mitotic figures, respectively, on a total of 500 cells of each category. Endometrial vascular proliferation was also verified by counting the number of Ki-67 positive cells on a total of 100 endothelial cells. The present study showed two proliferation peaks involving different cell groups. In the surface epithelium, the stroma, the blood vessels and the crypts, the highest labelling and mitotic indexes were noticed during proestrus, whereas for the basal glands these indexes significantly increased (P < 0.05) during estrus compared to late metestrus and anestrus. Furthermore, a slightly positive correlation (P < 0.05) was found between the labelling index in the basal glands and the serum progesterone levels, whereas the labelling indexes in the other cell groups were positively correlated with the estradiol-17 beta levels, although not always significantly. These findings suggest that regulation of the proliferation in the surface epithelium, the stroma, the blood vessels and the crypts is different from the proliferation in the basal glands.

Angiogenesis in canine mammary tumours: a morphometric and prognostic study.

Angiogenesis in canine mammary tumours (CMTs) has been described previously; however, only the intratumoural (IT) region has been studied and information on peritumoural (PT) angiogenesis is lacking. In this study, the blood vessel density (BVD), blood vessel perimeter (BVP) and blood vessel area (BVA) in IT and PT regions of 56 benign CMTs, 55 malignant CMTs and 13 samples of normal mammary gland tissue were analyzed. In addition, the blood endothelial cell proliferation (BECP) as an indicator of ongoing angiogenesis was investigated. The prognostic value of each parameter was also examined. Blood vessels and proliferating blood endothelial cells were present in IT and PT regions of both benign and malignant tumours. The vessels in the PT region had a significantly higher area and perimeter compared with those in the IT region. Malignant tumours showed significantly more vessels with a smaller total BVA and a higher BECP compared with benign tumours and control tissue. In the PT regions there was a significantly higher BVD, BVA and BVP compared with the vessels in control tissue. Only the IT and PT BVD and PT BECP in benign tumours allowed prediction of survival. The morphology of blood vessels in CMTs shows similarities with those in human breast cancer, which strengthens the case for the use of dogs with CMTs in comparative oncology trials.

Lymphangiogenesis in canine mammary tumours: a morphometric and prognostic study.

Canine mammary tumours (CMTs) are the most common tumours of entire female dogs and represent a promising model for human breast cancer. Little is known about the presence and prognostic value of lymphangiogenesis in CMTs. The aims of the present study were to analyze selected characteristics of lymphatic vessels in CMTs, to evaluate their prognostic significance and to compare these results with studies of human breast cancer. Fifty-six benign CMTs, 55 malignant CMTs and 13 control samples of normal canine mammary gland tissue were studied. Serial immunohistochemical labelling with the lymphatic marker prox-1 and the proliferation marker Ki67 was performed. In intratumoural (IT) and peritumoural (PT) regions, the lymphatic vessel density (LVD), mean lymphatic vessel perimeter (LVP) and relative area occupied by lymphatic vessels (LVA) were analyzed. Lymphatic endothelial cell proliferation (LECP) and tumour cell proliferation (TCP) were also measured. Lymphatic vessels were identified in IT and PT regions and lymphangiogenesis was present in both regions. The IT lymphatic vessels were smaller, less numerous and occupied a smaller relative area compared with those of the PT region. Although no differences in lymphatic vessel parameters were observed between benign and malignant tumours, control tissue differed significantly from neoplastic tissue. None of the lymphatic vessel parameters showed a prognostic value, except for LECP in PT regions of benign tumours. The findings were in accordance with results of investigations into human breast cancer, which supports the use of dogs with spontaneously occurring CMTs as an animal model in comparative oncology trials.

Age-related differences in mucosal barrier function and morphology of the small intestine in low and normal birth weight piglets.

To test the hypothesis that the mucosal maturation of the small intestine is altered in low birth weight piglets, pairs of naturally suckled low birth weight (LBW, n = 20) and normal birth weight (NBW, n = 20) littermate piglets were selected and sampled after 0, 3, 10, and 28 d of suckling. In vivo intestinal permeability was evaluated via a lactulose-mannitol absorption test. Other indirect measurements for mucosal barrier functioning included sampling for histology and immunohistochemistry (intestinal trefoil factor [ITF]), measuring intestinal alkaline phosphatase (IAP) activity, and immunoblotting for occludin, caspase-3, and proliferating cell nuclear antigen (PCNA). The lactulose-mannitol ratio did not differ between NBW and LBW piglets, but a significant increase in this ratio was observed in 28-d-old piglets (P = 0.001). Small intestinal villus height did not differ with age (P = 0.02) or birth weight (P = 0.20). In contrast, villus width (P = 0.02) and crypt depth (P < 0.05) increased gradually with age, but no birth-weight-related differences were observed. LBW piglets had significantly (P = 0.03) more ITF immunoreactive positive cells per villus area compared to NBW piglets, whereas no age (P = 0.82) or region-related (P = 0.13) differences could be observed. The activity of IAP in the small intestine was higher in newborn piglets compared to the older piglets. No significant differences in cell proliferation in the small intestine was observed (P = 0.47) between NBW and LBW piglets; the highest proliferation was seen in piglets of 28 d of age (P = 0.01). Newborn piglets had significantly fewer apoptotic cells, whereas more apoptotic cells were seen in piglets of 10 d of age (P < 0.01). In conclusion, birth weight did not affect the parameters related to intestinal barrier function investigated in this study, suggesting that the mucosal barrier function is not altered in LBW piglets. Nevertheless, these results confirm that the mucosal barrier function in the small intestine of piglets alters with age.

Supplementing formula-fed piglets with a low molecular weight fraction of bovine colostrum whey results in an improved intestinal barrier.

To test the hypothesis that a low molecular weight fraction of colostral whey could affect the morphology and barrier function of the small intestine, 30 3-d-old piglets (normal or low birth weight) were suckled (n = 5), artificially fed with milk formula (n = 5), or artificially fed with milk formula with a low molecular weight fraction of colostral whey (n = 5) until 10 d of age. The small intestine was sampled for histology (haematoxylin and eosin stain; anti-KI67 immunohistochemistry) and enzyme activities (aminopeptidase A, aminopeptidase N, dipeptidylpeptidase IV, lactase, maltase, and sucrase). In addition, intestinal permeability was evaluated via a dual sugar absorption test and via the measurement of occludin abundance. Artificially feeding of piglets reduced final BW (P < 0.001), villus height (P < 0.001), lactase (P < 0.001), and dipeptidylpeptidase IV activities (P < 0.07), whereas crypt depth (P < 0.001) was increased. No difference was observed with regard to the permeability measurements when comparing artificially fed with naturally suckling piglets. Supplementing piglets with the colostral whey fraction did not affect BW, enzyme activities, or the outcome of the dual sugar absorption test. On the contrary, the small intestines of supplemented piglets had even shorter villi (P = 0.001) than unsupplemented piglets and contained more occludin (P = 0.002). In conclusion, at 10 d of age, no differences regarding intestinal morphology and permeability measurements were observed between the 2 BW categories. In both weight categories, the colostral whey fraction affected the morphology of the small intestine but did not improve the growth performances or the in vivo permeability. These findings should be acknowledged when developing formulated milk for neonatal animals with the aim of improving the performance of low birth weight piglets.

Lymph drainage from the ovine tonsils: an anatomical study of the tonsillar lymph vessels.

Although the tonsils of sheep have gained much attention during the last decade, only few data are available on their lymph vessel architecture. Tonsillar lymph vessels are immunologically important as they form the efferent routes for locally activated immune cells to reach the draining lymph nodes. To gain insight into the tonsillar lymph drainage in the sheep, Indian ink and a casting polymer were injected into the interstitium of the five tonsils present in the heads of slaughtered sheep. This enabled us to determine the draining lymph node and to examine the microscopic organization of lymph vessels using light and scanning electron microscopy. No lymph vessels were observed within the tonsillar lymphoid follicles. The corrosion casts demonstrated that the lymphoid follicles are surrounded by numerous sacculated lymph sinuses that drain into a dense interfollicular lymph vessel network. From here, the lymph flows into single small lymph vessels that in turn drain into larger lymph vessels extending towards the medial retropharyngeal lymph node. The presented results can be valuable for immunological studies, for example during oral or intranasal vaccine development.