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.

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.

Spleen but not tumor infiltration by dendritic and T cells is increased by intravenous adenovirus-Flt3 ligand injection.

Dendritic cell (DC) expansion is regulated by the hematopoietic growth factor fms-like tyrosine kinase 3 ligand (Flt3L). DCs are critical to the control of tumor growth and metastasis, and there is a positive correlation between intratumoral DC infiltration and clinical outcome. In this report, we first demonstrate that single intravenous (i.v.) injections of adenovirus (Adv)-Flt3L significantly increased splenic dendritic, B, T and natural killer (NK) cell numbers in both normal and mammary tumor-bearing mice. In contrast, the numbers of DCs and T cells infiltrating the tumors were not increased. Consistent with the minimal effect on immune cell infiltration, i.v. Adv-Flt3L injections had no therapeutic activity against orthotopic mammary tumors. In addition, we noted tumor and Adv-Flt3L expansion of Gr1(+)CD11b(+) immature myeloid suppressor cells (IMSCs), which may inhibit the therapeutic efficacy of Adv-Flt3L-expanded DCs.

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.

Evaluation of multiple immune parameters after vaccination with modified live or killed bovine viral diarrhea virus vaccines.

The development of immunity to vaccine antigen was examined using three prime/boost strategies and the progression of immune activities was evaluated over the course of 8 weeks. Calves were vaccinated and multiple immune parameters were evaluated using several methods to assess humoral or cellular immunity from the same samples in parallel. The three vaccination protocols used were a killed vaccine followed by a killed boost (killed/killed), MLV vaccine and boost (MLV/MLV), or a MLV vaccine and killed boost (MLV/killed). All the vaccines used included modified live IBR/PI3 viruses to make the bystander context as similar as possible. The Singer strain of BVDV was used as the source antigen in the killed vaccine, and the NADL strain of BVDV was used in the MLV vaccine. Controls received a vaccine containing only MLV IBR/PI3. The assessment panel measured SN titers, as well as lymphocyte proliferation, cytokine mRNA expression, intracellular cytokine production, and released IFN-gamma after in vitro stimulation with three strains of BVDV virus. MLV/MLV and MLV/killed groups developed significant SN titers to the type 1 BVDV virus strains, Singer and NADL, and low crossover titers were also seen to the type 2 strain, 890 over the evaluation period. These two groups showed significant proliferation in response to the NADL virus as compared to controls. Multiple immune assessments were conducted simultaneously to attempt to provide a broader, more in depth evaluation of immune response to these BVDV vaccination protocols. We observed that the correlation among most of the assays conducted were weak; the correlation between SN titers and cellular proliferation assays demonstrated a moderate correlation.

Colostrum induced phenotypic and trafficking changes in maternal mononuclear cells in a peripheral blood leukocyte model for study of leukocyte transfer to the neonatal calf.

The relationship between the colostral environment and the function of leukocytes in colostrum is not clearly defined. This study examined the effects of defatted, acellular colostrum (AC) on the phenotype of peripheral blood mononuclear cells (PBMC) and their capacity to enter the circulation of neonatal calves after ingestion as a model of this relationship. Maternal PBMC were exposed to medium alone or medium supplemented with 25% AC. Expression of CD11a, CD11b, CD11c, CD43, CD49d, CD49e, and CD62L was assessed on freshly isolated and treated PBMC. Exposure to AC increased the percentage of cells expressing CD11a, CD11c and CD43, but decreased the percentage of cells expressing CD62L relative to freshly isolated PBMC. The density of expression of CD11b and CD11c was reduced, but increased for CD43 after exposure to AC relative to freshly isolated PBMC. Density of CD62L expression and percentage of cells expressing CD11a and CD43 were significantly different for cells treated with AC relative to medium alone. Further, these changes could not be attributed to occult bacterial contamination of the AC, as treatment of PBMC with LPS in the same medium yielded none of the observed changes. Maternal PBMC (treated as described) were labeled with the fluorescent tracer, PKH26-GL, and fed to neonatal calves within 6 h of birth. The circulation of these cells in the neonate was monitored by flow cytometry. We observed that: (1) cells exposed to AC, but not medium alone, entered the circulation; (2) peak trafficking occurred 12-24 h after ingestion; (3) a large fraction of labeled cells appeared in the neonatal circulation; and (4) labeled cells disappeared from circulation by 36 h after ingestion. This study indicates that exposure to the colostral environment induced phenotypic changes facilitating trafficking of colostral cells into the neonate.

Flt3 ligand bioactivity and pharmacology in neoplasia.

Fms-like tyrosine kinase 3 ligand (Flt3L) has multiple effects on the hematopoietic and immune systems. Further, preclinical studies have suggested potential therapeutic activity against cancer. Flt3L is a potent hematopoietic cytokine, capable of stimulating the expansion and differentiation of hematopoietic progenitor and stem cells. Administration of Flt3L mobilizes hematopoietic cells from the bone marrow (BM) into the blood, lymphoid organs, and parenchymal tissues. This mobilization activity, especially effective in combination with granulocyte colony stimulating factor (G-CSF), has stimulated studies of Flt3L in hematopoietic stem cell (HSC) transplantation. In addition to its effects on hematopoietic stem and progenitor cells, Flt3L has been shown to increase the frequency and number of dendritic cells (DCs) within the circulatory system and solid organs. DC expansion by Flt3L has been the focus of preclinical and clinical studies on antigen (Ag) specific T-cell mediated immunity. The mechanism for the augmentation of T-cell mediated immunity has yet to be completely identified, although Flt3L's ability to expand DCs in lymphoid and non-lymphoid tissues is involved. This expansion occurs primarily with DCs, which secrete interleukin (IL) 12. Consistent with the expansion of this DC population, treatment with Flt3L enhances T-cell mitogenesis and preferentially induces type 1 T-cell responses. However, the DCs resulting from Flt3L administration are immature, leading in some studies to the induction of tolerance. This review focuses on the effects of Flt3L on DCs and other effector populations, and on its potential activity as a therapeutic agent for cancer, alone and in combination with vaccines.

A region of tapasin that affects L(d) binding and assembly.

Tapasin has been shown to stabilize TAP and to link TAP to the MHC class I H chain. Evidence also has been presented that tapasin influences the loading of peptides onto MHC class I. To explore the relationship between the ability of tapasin to bind to TAP and the MHC class I H chain and the ability of tapasin to facilitate class I assembly, we have created novel tapasin mutants and expressed them in 721.220-L(d) cells. One mutant has a deletion of nine amino acid residues (tapasin Delta334-342), and the other has amino acid substitutions at positions 334 and 335. In this report we describe the ability of these mutants to interact with L(d) and their effects on L(d) surface expression. We found that tapasin Delta334-342 was unable to bind to the L(d) H chain, and yet it facilitated L(d) assembly and expression. Tapasin Delta334-342 was able to bind and stabilize TAP, suggesting that TAP stabilization may be important to the assembly of L(d). Tapasin mutant H334F/H335Y, unlike tapasin Delta334-342, bound to L(d). Expression of tapasin H334F/H335Y in 721.220-L(d) reduced the proportion of cell surface open forms of L(d) and retarded the migration of L(d) from the endoplasmic reticulum. In total, our results indicate that the 334-342 region of tapasin influences L(d) assembly and transport.