Expression of PC, PCK1, PCK2, LDHB, FBP1 and G6PC genes in the liver of cows in the transition from pregnancy to lactation*

The effect of the transition of dairy cows from pregnancy to lactation on the expression of genes encoding enzymes involved in hepatic glucose metabolism was studied. Six Holstein-Friesian heifers were used for this study. Liver samples were collected by biopsy on day 7 before expected parturition (-7) and days 3 and 21 after parturition (+3 and +21, respectively). The mRNA levels of pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase 1 (PCK1), phosphoenolpyruvate carboxykinase 2 (PCK2), lactate dehydrogenase B (LDHB), fructose-1,6-bisphosphatase 1 (FBP1) and glucose-6-phosphatase (G6PC) were measured using quantitative real-time PCR. The expression of PC and PCK2 mRNA on day 3 of lactation was significantly higher than that on day 7 before parturition (P<0.05) and slightly higher than on day 21 postpartum. The LDHB gene showed the highest expression level on day 3 of lactation, as compared with day 7 prepartum (P<0.001) and day 21 postpartum (P<0.001). No differences were shown in PCK1, FBP1 and G6PC expression levels between pregnancy and early lactation

NKT Cell Stimulation with α-Galactosylceramide Results in a Block of Th17 Differentiation after Intranasal Immunization in Mice

In a previous study we demonstrated that intranasal (i.n.) vaccination promotes a Th17 biased immune response. Here, we show that co-administration of a pegylated derivative of α-galactosylceramide (αGCPEG) with an antigen, even in the presence of Th17-polarizing compounds, results in a strong blocking of Th17 differentiation. Additional studies demonstrated that this phenomenon is specifically dependent on soluble factors, like IL-4 and IFNγ, which are produced by NKT cells. Even NK1.1 negative NKT cells, which by themselves produce IL-17A, are able to block Th17 differentiation. It follows that the use of αGCPEG as adjuvant would enable to tailor Th17 responses, according to the specific clinical needs. This knowledge expands our understanding of the role played by NKT cells in overall control of the cytokine microenvironment, as well as in the overall shaping of adaptive immune responses

Peritoneal Cavity Is Dominated by IFNγ-Secreting CXCR3 Th1 Cells.

The chemokine receptor CXCR3, which was shown to take part in many inflammatory processes, is considered as a Th1 specific marker. Here, we show in a mouse model that CXCR3 expressing CD4(+) cells preferentially migrate to the peritoneal cavity under steady-state conditions. The peritoneal cavity milieu leads to an up-regulated expression of CXCR3. However, blocking of known ligands of this chemokine receptor did not alter the preferential migration. The peritoneal cavity environment also results in an increased percentage of memory cells producing cytokines. Up-regulation of IFNγ production occurs mostly in CXCR3(+) cells considered as Th1, whereas the up-regulation of IL-4 affects mostly in CXCR3(-) cells which are considered as Th2. We conclude that the peritoneal cavity does not change the Th-lineage of the cells, but that domination of this anatomic niche by Th1 cells rather results from preferential migration to this compartment

Neutralization of CXCR3 ligands does not block preferential migration of CXCR3⁺ CD4⁺ T cells to the PerC.

<p>CXCR3⁺ and CXCR3⁻ CD4⁺ T cells isolated from donor animals were differentially labeled with CFSE or CMTMR, and mixtures containing equal numbers of cells were injected intravenously into naïve recipients pretreated with a cocktail of anti-CXCL9, anti-CXCL10 and anti-CXCL11 antibody or an appropriate isotype control. Eighteen hours after transfer, the ratio of transferred CXCR3⁺ cells to total transferred cells was determined in different organs by flow cytometry. Dots represent the ratio for individual animals. Horizontal lines indicate the mean for the group, and error bars correspond to the SEM. n.s. – not significant. Sp, spleen; PerC, peritoneal cavity.</p

The percentage of CXCR3⁺ CD4⁺ T cells is higher in PerC than in other organs.

<p>The percentage of CXCR3⁺ CD4⁺ T cells in different organs was determined by flow cytometry. (A) Cells isolated from naïve Balb/c animals were analyzed (n = 5). Each point represents an individual animal. (B) Cells isolated from 10 to 20 naïve Balb/c animals were pooled. Results from 8 independent experiments are presented. Error bars indicate SEM. cLN, cervical lymph node; mLN, mesenteric lymph node; PP, Peyer's patches; Sp, spleen; PerC, peritoneal cavity. (*) Statistically significant at P<0.05.</p

The PerC environment results in increased expression of CXCR3 on CD4⁺ T cells.

<p>(A) The expression level of CXCR3 on CD4⁺ cells from spleen (black line) or PerC (grey area) of BALB/c mice was determined by flow cytometry. (B) CXCR3⁺ and CXCR3⁻ CD4⁺ T cells were sorted from Sp of donor BALB/c mice and stained with CFSE. Cells were then adoptively transferred to naïve recipient animals by i.v or i.p. route. After 24 h, cells were re-isolated from spleens or peritoneal cavities and re-stained with an anti-CXCR3 antibody. The expression level of CXCR3 on CFSE⁺ cells was then analyzed by flow cytometry. The differences were statistically significant at P<0.001 (*), n.s. – not significant.</p

Peritoneal memory CD4⁺ T cells produce more cytokines than Sp derived cells.

<p>CXCR3⁺ CD62L^low CD44^high, CXCR3⁻ CD62L^low CD44^high and CXCR3⁻ CD62L^high CD44^low CD4⁺ T cells were sorted from Sp or PerC of C57BL/6 naïve (specific pathogen free) mice. Then, cells were re-stimulated with ionomycin and PMA and the expression of (A) IFNγ, (B) IL-4 and (C) IL-17 was measured by ELISA. Similar results were obtained in 2 independent experiments.</p