No abstract

Cyclic diguanylate (c-di-GMP) is a bacterial intracellular signaling molecule. We have shown that treatment with exogenous c-di-GMP inhibits Staphylococcus aureus infection in a mouse model. We now report that c-di-GMP is an immodulator and immunostimulatory molecule. Intramammary treatment of mice with c-di-GMP 12 and 6 h before S. aureus challenge gave a protective effect and a 10,000-fold reduction in CFUs in tissues (p < 0.001). Intramuscular vaccination of mice with c-di-GMP coinjected with S. aureus clumping factor A (ClfA) Ag produced serum with significantly higher anti-ClfA IgG Ab titers (p < 0.001) compared with ClfA alone. Intraperitoneal injection of mice with c-di-GMP activated monocyte and granulocyte recruitment. Human immature dendritic cells (DCs) cultured in the presence of c-di-GMP showed increased expression of costimulatory molecules CD80/CD86 and maturation marker CD83, increased MHC class II and cytokines and chemokines such as IL-12, IFN-γ, IL-8, MCP-1, IFN-γ-inducible protein 10, and RANTES, and altered expression of chemokine receptors including CCR1, CCR7, and CXCR4. c-di-GMP-matured DCs demonstrated enhanced T cell stimulatory activity. c-di-GMP activated p38 MAPK in human DCs and ERK phosphorylation in human macrophages. c-di-GMP is stable in human serum. We propose that cyclic dinucleotides like c-di-GMP can be used clinically in humans and animals as an immunomodulator, immune enhancer, immunotherapeutic, immunoprophylactic, or vaccine adjuvant.

Enzymes that hydrolyze complex carbohydrates play important roles in numerous biological processes that result in the maintenance of marine and terrestrial life. These enzymes often contain noncatalytic carbohydrate binding modules (CBMs) that have important substrate-targeting functions. In general, there is a tight correlation between the ligands recognized by bacterial CBMs and the substrate specificity of the appended catalytic modules. Through high-resolution structural studies, we demonstrate that the architecture of the ligand binding sites of 4 distinct family 35 CBMs (CBM35s), appended to 3 plant cell wall hydrolases and the exo-␤-D-glucosaminidase CsxA, which contributes to the detoxification and metabolism of an antibacterial fungal polysaccharide, is highly conserved and imparts specificity for glucuronic acid and/or ⌬4,5-anhydrogalaturonic acid (⌬4,5-GalA). ⌬4,5-GalA is released from pectin by the action of pectate lyases and as such acts as a signature molecule for plant cell wall degradation. Thus, the CBM35s appended to the 3 plant cell wall hydrolases, rather than targeting the substrates of the cognate catalytic modules, direct their appended enzymes to regions of the plant that are being actively degraded. Significantly, the CBM35 component of CsxA anchors the enzyme to the bacterial cell wall via its capacity to bind uronic acid sugars. This latter observation reveals an unusual mechanism for bacterial cell wall enzyme attachment. This report shows that the biological role of CBM35s is not dictated solely by their carbohydrate specificities but also by the context of their target ligands.carbohydrate protein binding ͉ enzyme targeting ͉ plant cell wall degradation ͉ protein cell attachment ͉ X-ray crystallography

Milk production is a function of the number and activity of mammary epithelial cells, regardless of stage of lactation. Milk yield is generally higher in multiparous cows than in primiparous cows, but persistency is usually greater in the latter group. We compared several measures related to metabolic activity, apoptosis, and endocrine control of mammary cell growth in 8 primiparous and 9 multiparous cows throughout lactation. Mammary gland biopsies were taken in early [10 d in milk (DIM)], peak (50 DIM), and late (250 DIM) lactation to evaluate gene expression and determine DNA and fatty acid synthase (FAS) content. Milk samples taken the day before the biopsies were used to detect protease activities and to determine stanniocalcin-1 (STC) concentrations. Blood samples served to measure insulin-like growth factor-1, prolactin, and STC concentrations. Milk yield was higher in multiparous cows than in primiparous cows at the 10 DIM (32.8 +/- 1.3 and 25.2 +/- 0.8 kg/d) and 50 DIM (38.0 +/- 1.2 and 29.8 +/- 1.1 kg/d), but it was the same for both groups at 250 DIM (23.9 +/- 1.5 and 23.8 +/- 1.1 kg/d). Except for stearoyl-coenzyme A desaturase, expression of genes related to milk synthesis was not affected by stage of lactation. However, gene expression of acetyl-coenzyme A carboxylase, beta-casein, and FAS was lower in early lactation in primiparous cows. Expression of both proapoptotic bax and antiapoptotic bcl-2 genes was higher in primiparous cows, whereas the bax-to-bcl-2 ratio was not changed. Mammary DNA concentration was higher in multiparous cows, as was the amount of FAS protein in early lactation. Two bands of protease activity were found in milk samples, and one of the bands had an apparent molecular weight similar to gelatinase A and was dependent on the stage of lactation. Serum insulin-like growth factor-1 increased with day of lactation and was higher in primiparous cows. Serum prolactin decreased in late lactation, but peak values were observed in early lactation for primiparous cows and peak lactation for multiparous cows. Milk STC content increased with advancing lactation. The results are consistent with a lower degree of differentiation and a greater capacity for cell renewal in the mammary gland of primiparous cows.

The transition from pregnancy to lactation is marked by metabolic, hormonal, and immunological changes that have an impact on the incidence of infectious and metabolic diseases. The aim of this study was to evaluate the effect on immune function and blood metabolite concentration of limiting milk production in early lactation to reduce negative energy balance. Twenty-two multiparous Holstein cows were milked either once a day (1x) or twice a day (2x) for the first week postpartum. All cows were milked twice daily for the rest of lactation. Blood concentrations of nonesterified fatty acids (NEFA), beta-hydroxybutyric acid (BHBA), calcium, bilirubin, urea, phosphorus, glucose, leptin, stanniocalcin-1, and 17beta-estradiol were determined in samples collected from 5 wk before scheduled calving to 5 wk after calving. Polymorphonuclear leukocytes (PMNL) were isolated from blood to conduct assays for chemotaxis, phagocytosis, and respiratory burst. Peripheral blood mononuclear cells (PBMC) were isolated to evaluate lymphocyte proliferation and cytokine production (tumor necrosis factor-alpha, IL-4, and interferon-gamma). Cows milked 1x produced 31% less milk than cows milked 2x during the first week of lactation. Over the following 13 wk of lactation, the milk production of cows milked 1x during the first week was 8.1% lower than for cows milked 2x. However, because the percentages of fat and protein were greater in the milk from 1x cows, the yields of milk components and energy-corrected milk were similar. Calving induced an increase in the concentrations of NEFA, BHBA, urea, and bilirubin. The increases in levels of NEFA and BHBA were greater in cows milked 2x than in cows milked 1x. During the same period, the serum glucose concentration decreased but remained greater in cows milked 1x. Serum calcium on d 4 and serum phosphorus on d 4 and 5 were greater in cows milked 1x. The differences between the 2 groups persisted beyond treatment until postpartum d 24 for NEFA and glucose and until postpartum d 14 for BHBA. After calving, the concentrations of leptin and stanniocalcin-1 decreased. During the first week postpartum, the decrease of leptin was less marked in cows milked 1x. The immune functions of PBMC and PMNL isolated from experimental cows and incubated using a standard medium did not show clear-cut peripartum immunosuppression. These variables were not significantly affected by the treatments, with the exception of interferon-gamma secretion, which was greater on d 5 and 14 in cows milked 1x. In conclusion, limiting milk production in early lactation had positive effects on metabolite concentration, but larger studies are necessary to establish if this could reduce disease incidence.