Adrian J. Reber

Neonatal Immune Development in the Calf and Its Impact on Vaccine Response

In this article we cover the immunologic response as it develops, the components of passive immunity, and the immune response of young calves. We discuss interference from maternal immunity in the development of specific immunity and vaccine strategies for developing protection against pathogens in calves.

Transfer of maternal colostral leukocytes promotes development of the neonatal immune system Part II. Effects on neonatal lymphocytes

It has been established that maternal leukocytes, conditioned by the mammary environment, cross the neonatal gut and circulate in the newborn calf. However, the impact of these cells on the development of neonatal immunity remains to be determined. This study examined the effects of maternal colostral leukocytes on development and maturation of neonatal adaptive immunity by examining the expression of surface markers on neonatal lymphocytes. At birth, neonatal calves were fed whole colostrum, or colostrum that had the maternal cells removed (cell-free colostrum), from their respective dams. Peripheral blood samples were collected at regular intervals over the first 4 weeks of life and lymphocytes were evaluated for surface expression of cellular markers. The results of these studies demonstrated that calves receiving whole colostrum had fewer CD11a positive lymphocytes in circulation during the first 2 weeks of life and this marker was expressed at a lower density than calves receiving cell-free colostrum. In addition, calves receiving whole colostrum also had a higher percentage of lymphocytes expressing the activation markers CD25 and CD26 by 7 days after birth. During the first week of life, lymphocytes from calves receiving whole colostrum had a higher density of MHC class I expression on their surfaces than cells from calves receiving cell-free colostrum. In general, these results indicate that transfer of maternal cells with colostrum allows for more rapid development of lymphocytes and maternal cells appeared to enhance their activation.

Transfer of maternal colostral leukocytes promotes development of the neonatal immune system: I. Effects on monocyte lineage cells

Although it has been established that maternal leukocytes traffic from colostrum into the neonatal circulation, the effects of these cells on neonatal immunity are only beginning to be understood. This study examined the effects of maternal colostral leukocytes on development and maturation of neonatal antigen presenting cells. At birth, groups of neonatal calves received whole or cell-free colostrum (CFC) from their respective dams. Peripheral blood samples were obtained over the first 4 weeks of life, and expression of surface markers associated with cellular activation and physiological stress were monitored on monocyte lineage cells. Calves receiving cell-free colostrum at birth expressed elevated levels of CD11a, CD11c, and CD14, compared to calves receiving whole colostrum (C). Calves receiving cell-free colostrum had an elevated number of monocytes in the peripheral blood during the first 2 weeks of life, however, these cells expressed lower levels of expression of CD25 and MHC class I compared to calves receiving whole colostrum. The most significant differences in marker expression occurred within the first 7 days of life.

Rapid infusion of a phospholipid emulsion attenuates the effects of endotoxaemia in horses

REASONS FOR PERFORMING STUDY Endotoxaemia currently is associated with a poor prognosis in horses. The results of recent trials in other species indicate that phospholipid emulsions reduce the deleterious effects of endotoxin (LPS). However, in a previous study in horses, a 2 h infusion of emulsion caused an unacceptable degree of haemolysis. HYPOTHESIS Rapid administration of a lower total dose of emulsion would reduce the effects of LPS and induce less haemolysis; the emulsion would reduce inflammatory effects of LPS in vitro. METHODS Twelve healthy horses received an i.v. infusion either of saline or a phospholipid emulsion (100 mg/kg), followed immediately by E. coli 055:B5 LPS (30 ng/kg). Clinical parameters, haematological profiles, serum tumour necrosis factor (TNF) activity, serum lipid profiles, urine analyses and severity of haemolysis were monitored before and at selected times after LPS. Monocytes were also incubated in vitro with LPS in the presence or absence of emulsion, after which TNF and tissue factor activities were determined. RESULTS Clinical signs of endotoxaemia were reduced in horses receiving the emulsion, including clinical score, heart rate, rectal temperature, serum TNF activity, and the characteristic leucopenic response to LPS, when compared to horses not receiving the emulsion. Three horses receiving the emulsion had none, 2 had mild and one had moderate haemolysis. There were no differences in urinalysis results and creatinine concentrations, either within the groups over time or between the groups. Serum concentrations of phosphatidylcholine, bile acids and triglycerides peaked immediately after the infusion; there were no significant changes in concentrations of nonesterified fatty acids or cholesterol. Incubation of equine monocytes with emulsion prevented LPS-induced TNF and tissue factor activities. CONCLUSIONS Rapid administration of emulsion significantly reduced inflammatory effects of LPS in vivo and caused a clinically insignificant degree of haemolysis. The results of the in vitro studies indicate that emulsion prevents not only LPS-induced synthesis of cytokines, but also expression of membrane-associated mediators (i.e. tissue factor). POTENTIAL RELEVANCE Rapid i.v. administration of emulsions containing phospholipids that bind endotoxin may provide a clinically useful method of treating endotoxaemia in horses.

Targeting the PI3K and MAPK pathways to treat Kaposi’s sarcoma-associated herpes virus infection and pathogenesis

Cells require the ability to appropriately respond to signals in their extracellular environment. To initiate, inhibit and control these processes, the cell has developed a complex network of signaling cascades. The phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways regulate several responses including mitosis, apoptosis, motility, proliferation, differentiation and many others. It is not surprising, therefore, that many viruses target the PI3K and MAPK pathways as a means to manipulate cellular function. Recently, Kaposi’s sarcoma-associated herpes virus (KSHV) has been added to the list. KSHV manipulates the PI3K and MAPK pathways to control such divergent processes as cell survival, cellular migration, immune responses, and to control its own reactivation and lytic replication. Manipulation of the PI3K and MAPK pathways also plays a role in malignant transformation. Here, the authors review the potential to target the PI3K and MAPK signaling pathways to inhibit KSHV infection and pathogenesis.

Effects of antifungal drugs and delivery vehicles on morphology and proliferation of equine corneal keratocytes in vitro

OBJECTIVE To evaluate the effects of topical antifungal drugs and delivery vehicles on the morphology and proliferation rate of cultured equine keratocytes. STUDY POPULATION 16 corneas obtained from 8 apparently ophthalmologically normal horses < 0.5 hours after euthanasia for reasons unrelated to the study. PROCEDURES Primary cultures of equine keratocytes were obtained from corneal stroma and were exposed to several concentrations of 3 commonly used, topically applied antifungals: natamycin, itraconazole, and miconazole. In addition, effects of drug delivery vehicles DMSO, benzalkonium chloride, and carboxymethylcellulose and a combination vehicle composed of polyethylene glycol, methylparaben, and propylparaben were also evaluated. Morphological changes and cellular proliferation were assessed 24, 48, and 72 hours after application. RESULTS At the highest concentrations tested, all antifungals caused marked cellular morphological changes and inhibited proliferation. At low concentrations, natamycin and miconazole induced rounding, shrinking, and detaching of the cells with inhibition of cellular proliferation. Natamycin caused the most severe cellular changes. Itraconazole, at the low concentrations, caused minimal morphological changes and had a minimal effect on proliferation. All vehicles tested had significantly less effects on cellular morphology and proliferation when compared with the antifungals, except for the combination vehicle, which caused severe morphological changes and inhibited proliferation, even at low concentrations. The DMSO had minimal effects on cellular morphology and proliferation, even at high concentrations. CONCLUSIONS AND CLINICAL RELEVANCE Itraconazole had significantly less cytotoxic effects on equine keratocytes in culture than did natamycin or miconazole. Natamycin had severe cytotoxic effects in vitro.

AIDS related viruses, their association with leukemia, and Raf signaling.

Leukemia is characterized by the production of an excessive number of abnormal white blood cells. Over time, this expanding population of poorly/non- functional white blood cells overwhelms the normal function of the bodys blood and immune systems. DNA translocations have been found common to leukemia, including Raf mutations. While the cause of leukemia is not known, several risk factors have been identified. In this review, we present an update on the role of AIDS related viruses as an etiology for leukemia. Human immunodeficiency virus-1 and -2 (HIV-1; -2) are the cause for the development of acquired immune deficiency syndrome (AIDS). Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), Human papillomavirus (HPV), and Kaposis sarcoma-associated herpesvirus (KSHV) are specifically implicated in AIDS associated malignancies. However, there are other viruses that are associated to a lesser extent with the AIDS condition and they are Human T-cell leukemia virus-1 (HTLV-1), hepatitis B virus (HBV), hepatitis C virus (HCV), and human herpesvirus-6 (HHV-6). Of these viruses, HTLV-1 has been etiologically associated with leukemia. Recent evidence suggests that EBV, HBV, HCV, and KSHV may also play a role in the development of some types of leukemia. Raf signaling has been shown to aid in the infection and pathogenesis of many of these viruses, making Raf pathway components good potential targets for the treatment of leukemia induced by AIDS related viruses.

Effect of invariant chain on major histocompatibility complex class I molecule expression and stability on human breast tumor cell lines

Invariant chain (Ii) binds to the human leukocyte antigen (HLA) class II molecule and assists it in the process of peptide acquisition. In addition, Ii binds to the HLA class I molecule, although there has been little study of its effects on the HLA class I molecule. In addition to its normal expression on antigen-presenting cells, Ii expression is up regulated in a variety of tumors. By flow cytometric analysis, we found that expression of Ii resulted in an increase in the number of cell surface HLA class I molecules and in the proportion of unstable HLA class I molecules at the surface of breast tumor cell lines. These data suggest that the expression of Ii by tumor cells may quantitatively and qualitatively alter the presentation of antigens on those cells.

Establishing a reproducible method for the culture of primary equine corneal cells.

OBJECTIVE To establish a reproducible method for the culture of primary equine corneal epithelial cells, keratocytes, and endothelial cells and to describe each cells morphologic characteristics, immunocytochemical staining properties and conditions required for cryopreservation. PROCEDURES Corneas from eight horses recently euthanized for reasons unrelated to this study were collected aseptically and enzymatically separated into three individual layers for cell isolation. The cells were plated, grown in culture, and continued for several passages. Each cell type was characterized by morphology and immunocytochemical staining. RESULTS All three equine corneal cell types were successfully grown in culture. Cultured corneal endothelial cells were large, hexagonal cells with a moderate growth rate. Keratocytes were small, spindloid cells that grew rapidly. Epithelial cells had heterogeneous morphology and grew slowly. The endothelial cells and keratocytes stained positive for vimentin and were morphologically distinguishable from one another. The epithelial cells stained positive for cytokeratin. Keratocytes and endothelial cells were able to be cryopreserved and recovered. The cryopreserved cells maintained their morphological and immunocytochemical features after cryopreservation and recovery. DISCUSSION This work establishes reproducible methods for isolation and culture of equine corneal keratocytes and endothelial cells. Cell morphology and cytoskeletal element expression for equine corneal epithelial cells, keratocytes, and endothelial cells are also described. This has not previously been reported for equine corneal cells. This report also demonstrates the ability to preserve equine keratocytes and endothelial cells for extended periods of time and utilize them long after the primary-cell collection, a feature that has not been reported for veterinary corneal cell culture.