Donald H. Schlafer

Abnormal Expression of Trophoblast Major Histocompatibility Complex Class I Antigens in Cloned Bovine Pregnancies Is Associated with a Pronounced Endometrial Lymphocytic Response

Abstract Early embryonic losses are much higher in nuclear transfer (cloned) pregnancies, and this is a major impediment to improving the efficiency of cloned animal production. In cattle, many of these losses occur around the time of placental attachment from the fourth week of gestation. We studied the potential for altered immunologic status of cloned pregnancies to be a contributing factor to these embryonic losses. Expression of major histocompatibility complex class I (MHC-I) by trophoblast cells and distribution of endometrial T-lymphocyte numbers were investigated. Six 5-wk-old cloned pregnancies were generated, and 2 others at 7 and 9 wk were also included, all derived from the same fetal cell line. All 8 cloned placentas displayed trophoblast MHC-I expression. None of the 8 controls (4–7 wk old) showed any MHC-I expression. The percentage of trophoblast cells expressing MHC-I varied in the clones from 17.9% to 56.5%. Numbers of T lymphocytes (CD3+ lymphocytes) were significantly higher in the endometrium of the majority of cloned pregnancies compared with controls. In the cloned pregnancies, large aggregates of T cells were frequently observed in the endometrium in addition to increased numbers of diffusely spread subepithelial lymphocytes. As trophoblast MHC-I expression is normally suppressed during early gestation, the observed MHC-I expression in the cloned pregnancies is likely to have induced a maternal lymphocytic response that would be detrimental to maintaining viability of the cloned pregnancy. These findings support a role for immunologic rejection in the syndrome of early embryonic loss in cloned bovine pregnancies.

Chronic in vivo imaging in the mouse spinal cord using an implanted chamber

Understanding and treatment of spinal cord pathology is limited in part by a lack of time-lapse in vivo imaging strategies at the cellular level. We developed a chronically implanted spinal chamber and surgical procedure suitable for time-lapse in vivo multiphoton microscopy of mouse spinal cord without the need for repeat surgical procedures. We routinely imaged mice repeatedly for more than 5 weeks postoperatively with up to ten separate imaging sessions and observed neither motor-function deficit nor neuropathology in the spinal cord as a result of chamber implantation. Using this chamber we quantified microglia and afferent axon dynamics after a laser-induced spinal cord lesion and observed massive microglia infiltration within 1 d along with a heterogeneous dieback of axon stumps. By enabling chronic imaging studies over timescales ranging from minutes to months, our method offers an ideal platform for understanding cellular dynamics in response to injury and therapeutic interventions.

The bovine placenta before and after birth: placental development and function in health and disease.

This paper reviews bovine placental development, anatomy (microscopic and gross), nomenclature and classification. The paper focuses on the biology of those specialized cells that arise from the outermost layer of very early embryos, the trophoblast cells, and on placental macrophages, cells that play a key role in fetal/placental defense. Data is presented from an immunohistochemical quantitative study that characterizes the ontogeny of placental macrophages using placental tissues from 21 cows (sampled from 4 months of pregnancy through the post partum period). Understanding of bovine placental development is essential for veterinarians, pathologists, diagnosticians and researchers. Lesions of diagnostic significance can be recognized for many economically important infectious abortifacient diseases, and there is growing evidence that pregnancy failure of cloned calves is due in part to unexplained placental failure. Placentology and placental pathology are becoming of increasing importance.

Evidence for expression of both classical and non-classical major histocompatibility complex class I genes in bovine trophoblast cells

During the third trimester of pregnancy bovine trophoblast cells in the interplacentomal and arcade regions of the placenta express major histocompatibility complex class I (MHC‐I) antigens. At parturition immunological recognition of MHC‐I antigens appears to contribute to normal placental release. Therefore, we hypothesized that during late pregnancy bovine trophoblast cells express polymorphic, classical MHC‐I antigens.

A Single Base Pair Mutation Encoding a Premature Stop Codon in the MIS Type II Receptor Is Responsible for Canine Persistent Müllerian Duct Syndrome

Müllerian inhibiting substance (MIS), a secreted glycoprotein in the transforming growth factor-beta family of growth factors, mediates regression of the Müllerian ducts during embryonic sex differentiation in males. In persistent Müllerian duct syndrome (PMDS), rather than undergoing involution, the Müllerian ducts persist in males, giving rise to the uterus, fallopian tubes, and upper vagina. Genetic defects in MIS or its receptor (MISRII) have been identified in patients with PMDS. The phenotype in the canine model of PMDS derived from the miniature schnauzer breed is strikingly similar to that of human patients. In this model, PMDS is inherited as a sex-limited autosomal recessive trait. Previous studies indicated that a defect in the MIS receptor or its downstream signaling pathway was likely to be causative of the canine syndrome. In this study, the canine PMDS phenotype and clinical sequelae are described in detail. Affected and unaffected members of this pedigree are genotyped, identifying a single base pair substitution in MISRII that introduces a stop codon in exon 3. The homozygous mutation terminates translation at 80 amino acids, eliminating much of the extracellular domain and the entire transmembrane and intracellular signaling domains. Findings in this model could enable insights to be garnered from correlation of detailed clinical descriptions with molecular defects, which are not otherwise possible in the human syndrome.

Rotaviral and Coronaviral Diarrhea

Rotaviruses and coronaviruses are very common causes of calfhood diarrhea throughout the world. Rotaviral and coronaviral infections in neonatal calves can produce a severe diarrhea of high morbidity but of variable mortality depending upon secondary bacterial infections and electrolyte imbalances. Laboratory diagnosis of rotaviral and coronaviral diarrhea is mostly done by detection of virus particles or virus antigens in fecal specimens. The sample of choice for these diagnostic tests is a fresh fecal sample collected directly from the calf as close as possible to the onset of diarrhea.

Intrauterine growth retardation and the circulatory responses to acute hypoxemia in fetal sheep

Intrauterine growth retardation has been produced experimentally by umbilical placental embolization for 9 days (early intrauterine growth retardation) in pregnant sheep. Fetuses with early intrauterine growth retardation had a 20% decrease in mean body weight and 33% decrease in placental blood flow. However, the regional blood flow distribution was not significantly different at rest between the embolized and normally grown fetuses despite the 39% decrease in fetal arterial oxygen content. The purpose of this study was to determine the circulatory responses to acute hypoxemic stress in the early development of intrauterine growth retardation. We found that the regional blood flow distribution was not significantly different during imposed acute hypoxemia between the seven fetuses with early intrauterine growth retardation and seven nonembolized normally grown fetuses. We conclude that growth-retarded fetuses are able to meet basal metabolic oxygen requirements and to respond normally to imposed acute hypoxemia until the placental circulatory reserve capacity is depleted.

Diagnosis of Borrelia‐associated uveitis in two horses

Borrelia burgdorferi, the etiologic agent of Lyme disease is a tick born spirochetal infection. Clinical signs of Lyme borreliosis are uncommon in horses, but when present they are often vague and nonspecific. In horses, Lyme borreliosis has been implicated in musculoskeletal, neurological, reproductive, and ocular disorders, including uveitis, but definitive diagnosis can be challenging as the causative agent is rarely isolated and serologic tests can be unreliable and do not confirm active disease. Here, we report two cases of equine uveitis associated with B. burgdorferi based on the identification of spirochetes within ocular fluids and confirmed with PCR testing. The two cases illustrate some of the challenges encountered in the recognition and diagnosis of equine Lyme borreliosis. Although only one of many possible causes of equine uveitis, Lyme disease should be considered a differential diagnosis, especially in endemic areas. Given the possibility for false negative results of serum tests during uveitis associated with B. burgdorferi and the failure of such tests to confirm active infection, a combination of cytologic assessment, antibody, and/or PCR testing of ocular fluids may be worthwhile if the clinical suspicion for Lyme uveitis is high.

Endotoxemia in pregnant cows: Comparisons of maternal and fetal effects utilizing the chronically catheterized fetus

We utilized the chronically catheterized bovine fetus to compare maternal and fetal responses to maternal lipopolysaccharide (LPS) infusion. Our hypothesis was that LPS-induced abortion was primarily a maternal luteolytic event with minimal transplacental fetal exposure. Fetal tibial arteries, amniotic, allantoic cavities and maternal carotid arteries were catheterized. Three cows had patent catheters with viable fetuses (190 to 200 days of gestation) 1 week after operation and were included in the study. Following a 2-day maternal and fetal baseline, 0.5 mug Salmonella typhimurium LPS/kg was infused into a maternal jugular vein over a 2-hour period. Maternal and fetal responses were monitored clinically, biochemically and hormonally. The maternal response consisted of marked increases in plasma prostaglandin F(2alpha) metabolite (PGFM), tumor necrosis factor (TNF), ACTH and cortisol with a dramatic maternal leucopenia within 2 hours. Progesterone concentrations decreased within 7 hours (P<0.05). The LPS was rapidly cleared from maternal circulation and no transplacental exposure was detected in the fetuses. Fetal responses to maternal endotoxemia consisted of increased ACTH and cortisol concentrations with delayed increases in PGE(2); TNF did not change in fetal fluids following maternal endotoxemia. There was a fetal leucocytosis within 2 hours. The results indicate that the fetus does not appear to play a major role in the pathogenesis of LPS-induced abortions. However, the role of maternal TNF in endotoxin abortion requires further study.

Ontogeny of inflammatory cell responsiveness: superoxide anion generation by phorbol ester-stimulated fetal, neonatal, and adult bovine neutrophils.

Newborn calves, like human infants, are uniquely susceptible to bacterial infections. Part of this increased susceptibility may be related to defects in newborn polymorphonuclear leukocyte (PMN) defensive functions. It remains unclear whether reported deficits in newborn PMN function represent maturational disorders or are manifestations of some form of perinatal suppression phenomenon. We therefore compared the ability of bovine newborn PMNs (less than 24 h old), newborn PMNs (7–10 days of age), fetal PMNs (210–220 days gestational age), and adult PMNs to generate superoxide anion (O2−) as an indicator of respiratory burst activity. Citrated biood was collected, and PMNs were isolated to greater than 95% purity and 98% viability. O2− generation was measured as the superoxide dismutase-inhibitable (10 μg/ml) reduction of ferricytochrome c (2 mg/ml) after activation of PMNs with phorbol myristate acetate (PMA, 2 μg/ml) to directly stimulate protein kinase C. The reaction kinetics were measured (37°C, 550 nm) using a spectrophotometer and chart recorder for continuous monitoring. O2− generation was measured for 5 min after the initial lag period and the total nanomoles of O2− generated calculated using the extinction coefficient for ferricytochromec. Newborn PMNs (N=10) generated significantly less O2− (5.7 ±0.8 nmol O2−/106 cells/5 min,P < 0.01) than did adult PMNs (N=14) (9.6 ±2.1 nmol O2−/1010 cells/5 min) or fetal PMNs (N=4) (10.7 ±0.7 nmol O2−/106 cells/5 min). PMNs from 7-to 10-day-old calves (N=9) generated almost identical amounts of O2− as newborn PMNs (5.7 ±1.6 nmol O2−/106 ceils/5 min). There was no difference in measured lag time period between new-born and adult PMNs, but fetal PMNs had significantly reduced (P < 0.01) mean lag time. The data indicated that bovine newborn PMNs have a decreased ability to generate O2− in response to PMA stimulation, which persists for at least 7–10 days, and that this functional decrement may be a manifestation of some form of perinatal PMN suppression phenomenon rather than a developmental abnormality since fetal PMNs produced O2− as well as adult PMNs.