Effect of the presence of the mammary gland on periparturient immunosuppression in dairy cows

Impaired neutrophil and lymphocyte function during the periparturient period is a contributing factor to the high incidence of infectious disease observed in the periparturient cow. Using 8 multiparous Jersey cows, we demonstrated that populations of T cells, especially T helper cells and gd T cells declined during the periparturient period coincident with reported losses of immune cell function;We hypothesized that milk production may be an important immunosuppressive factor. To test this hypothesis, we used 10 mastectomized and 8 intact multiparous Jersey cows and examined phenotype of peripheral blood mononuclear cells (PBMC), adhesion molecule expression and myeloperoxidase activity in neutrophils, and plasma level of steroid hormones. In intact cows, all T cell subset populations (CD3, CD4, CD8 and gd T cell receptor positive cells) showed significant declines toward parturition, and monocyte percentage increased significantly at parturition. These changes were significantly different from mastectomized cows. Mastectomy eliminated almost all changes in leukocyte subset proportions seen around parturition;Expression of leukocyte adhesion molecules was different in intact and mastectomized cows. Expression of b 2-integrins in intact cows was highest at parturition, and this expression was greater in intact cows than in mastectomized cows throughout the study. L-selectin expression exhibited a sudden decrease at parturition with recovery within a day after parturition in both intact and mastectomized cows. The ability of neutrophils to kill microbes as assessed by neutrophil myeloperoxidase activity decreased before parturition in both groups. While there was a quick recovery of neutrophil myeloperoxidase activity in mastectomized cows, there was no recovery in intact cows after parturition throughout the study which lasted until d 20 post partum. Milk production seems to exacerbate periparturient immunosuppression, especially with regard to recovery of neutrophil myeloperoxidase activity;Steroid hormone (progesterone, estrone, estradiol, and cortisol) analysis showed that mastectomy did not decrease the level of steroid hormones but rather increased estrogen level. Increase in steroid hormones do not seem to contribute to the periparturient immunosuppression in PBMC, although they may have certain effects on neutrophil function and L-selectin expression, especially before parturition

Innate immune response to intramammary infection with Serratia marcescens and Streptococcus uberis

Streptococcus uberis and Serratia marcescens are Gram-positive and Gram-negative bacteria, respectively, that induce clinical mastitis. Once initial host barrier systems have been breached by these pathogens, the innate immune system provides the next level of defense against these infectious agents. The innate immune response is characterized by the induction of pro-inflammatory cytokines, as well as increases in other accessory proteins that facilitate host recognition and elimination of the pathogens. The objective of the current study was to characterize the innate immune response during clinical mastitis elicited by these two important, yet less well-studied, Gram-positive and Gram-negative organisms. The pro-inflammatory cytokine response and changes in the levels of the innate immune accessory recognition proteins, soluble CD14 (sCD14) and lipopolysaccharide (LPS)-binding protein (LBP), were studied. Decreased milk output, induction of a febrile response, and increased acute phase synthesis of LBP were all characteristic of the systemic response to intramammary infection with either organism. Infection with either bacteria similarly resulted in increased milk levels of IL-1$\beta$, IL-8, IL-10, IL-12, IFN-$\gamma$, TNF-$\alpha$, sCD14, LBP, and the complement component, C5a. However, the duration of and/or maximal changes in the increased levels of these inflammatory markers were significantly different for several of the inflammatory parameters assayed. In particular, S. uberis infection was characterized by the sustained elevation of higher milk levels of IL-1$\beta$, IL-10, IL-12, IFN-$\gamma$, and C5a, relative to S. marcescens infection. Together, these data demonstrate the variability of the innate immune response to two distinct mastitis pathogens

Three-dimensional alignment of cellulose II microcrystals under a strong magnetic field

In this study, enzymatic synthesis was conducted using cellodextrin phosphorylase (CDP), sucrose phosphorylase (SP), and sucrose with 1-azido-1-deoxy-β-glucoside (β-glucosyl azide) as the acceptor in phosphate buffer at pH 7.0. This yielded cellulose oligomers (degree of polymerization, DP ≈ 10) with azido groups at the reducing end as a white precipitate. A suspension of cellulose microcrystals with exposed azido groups on the surface was obtained via dissolution and recrystallization of the synthetic products dispersed in water by heating. The flat, ribbon-like cellulose microcrystals were a crystalline form of cellulose II and were several micrometers in length and several hundred nanometers in width. The microcrystals were 5.1–5.2 nm thick, which is equivalent to the chain length of cellulose oligomers with DP ≈ 10. When the cellulose II microcrystal suspensions were dried under a horizontal static magnetic field of 8 T, oriented films were obtained, wherein the microcrystals were aligned three-dimensionally. Synchrotron X-ray diffraction studies of the films revealed that the easy and intermediate axes (χ₁ and χ₂, respectively) of the cellulose II crystals corresponded approximately to the [1 1 0] and [1 ̅₁ 0] directions, respectively

Titanium dioxide nanoparticles enhance mortality of fish exposed to bacterial pathogens

Nano-TiO2 is immunotoxic to fish and reduces the bactericidal function of fish neutrophils. Here, fathead minnows (Pimephales promelas) were exposed to low and high environmentally relevant concentration of nano-TiO2 (2 ng g−1 and 10 μg g−1 body weight, respectively), and were challenged with common fish bacterial pathogens, Aeromonas hydrophila or Edwardsiella ictaluri. Pre-exposure to nano-TiO2 significantly increased fish mortality during bacterial challenge. Nano-TiO2 concentrated in the kidney and spleen. Phagocytosis assay demonstrated that nano-TiO2 has the ability to diminish neutrophil phagocytosis of A. hydrophila. Fish injected with TiO2 nanoparticles displayed significant histopathology when compared to control fish. The interplay between nanoparticle exposure, immune system, histopathology, and infectious disease pathogenesis in any animal model has not been described before. By modulating fish immune responses and interfering with resistance to bacterial pathogens, manufactured nano-TiO2 has the potential to affect fish survival in a disease outbreak

Functional relationships between cyclodextrin glucanotransferase from an alkalophilic Bacillus and α-amylases Site-directed mutagenesis of the conserved two Asp and one Glu residues

AbstractComparison of the amino acid sequences of cyclodextrin glucanotransferases (CGTases) with those of α-amylases revealed that two Asp and one Glu residues, which are considered to be the catalytic residues in α-amylases, were also conserved in CGTases. To analyze the function of the three conserved amino acid residues in CGTases, site-directed mutagenesis was carried out. The three mutant CGTases, in which Asp229, Glu257 and Asp328 were individually replaced by Asn or Gln, completely lost both their starch-degrading and β-cyclodextrin-forming activities, whereas another mutant CGTase, in which Glu264 replaced by Gln, retained these activities. The three inactive enzymes retained the ability to be bound to starch. These results suggest that Asp229, Glu257 and Asp328 play an important role in the enzymatic reaction catalyzed by CGTase and that a similar catalytic mechanism is present in both CGTases and α-amylases

Canine Peripheral Blood Lymphocyte Phenotyping by 7-Color Multiparameter Flow Cytometry

Objective: To characterize baseline canine lymphocyte phenotypes including lymphocytes coexpressing multiple markers by novel 7-color multiparameter flow cytometry. Study Design: Fresh canine peripheral blood lymphocytes of 79 healthy 26-week-old Beagle or Beagle-mix dogs were stained and analyzed. Results: The high number of samples and acquired flow data (averaging 1.9x105 cells/sample) allowed the detection of minor lymphocyte subsets coexpressing multiple lymphocyte markers. The averaged percentages of major lymphocyte subsets of CD3+, CD4+, CD8+, CD21+ and gd TCR+ cells from this study were 74.0, 43.6, 14.3, 9.6, and 0.2, respectively, which were comparable but uniquely different from other reports as they were simultaneously detected in the same sample. We demonstrated that the commonly used CD21 and CD3 monoclonal antibody (mAb) clones, previously recommended not to be used in the same staining, could and should be used together with the proper steps of lymphocyte gating. We found a high percentage (10.3%) of unidentified CD21–CD3+CD4–CD8–gdTCR– lymphocyte subset that has never been reported. The intensive gating strategy and the mean percentages of each lymphocyte subset to their parent subsets and to the total lymphocyte population are presented and discussed. Conclusion: The canine lymphocyte phenotypes were fully characterized. This novel multiparameter flow cytometry method is a powerful approach to in-crease the accuracy of lymphocyte phenotyping in dogs

In Vitro Synthesis of Branchless Linear (1 → 6)-α-d-Glucan by Glucosyltransferase K: Mechanical and Swelling Properties of Its Hydrogels Crosslinked with Diglycidyl Ethers

A hydrogel was prepared from a polysaccharide, enzymatically synthesized through a one-pot reaction in aqueous solution, and its properties as a functional material were evaluated. Enzymatic synthesis using glucosyltransferase K obtained from Streptococcus salivarius ATCC 25975 was performed with sucrose as a substrate. The synthetic product was unbranched linear (1 → 6)-α-d-glucan with a high molecular weight, Mw: 1.0–3.0 × 105. The synthesized (1 → 6)-α-d-glucan was insoluble in water and crystallized in a monoclinic unit cell, which is consistent with the hydrated form of dextran. Transparent and highly swellable (1 → 6)-α-d-glucan hydrogels were obtained by crosslinking with diglycidyl ethers. The hydrogels showed no syneresis and no volume change during compression, resulting in the retention of shape under repeated compression. The elastic moduli of these hydrogels (<60 kPa) are smaller than those of other polysaccharide-based hydrogels having the same solid contents. The oven-dried gels could be restored to the hydrogel state with the original transparency and a recovery ratio greater than 98%. The mechanism of water diffusion into the hydrogel was investigated using the kinetic equation of Peppas. The properties of the hydrogel are impressive relative to those of other polysaccharide-based hydrogels, suggesting its potential as a functional biomaterial

Comparison of Humoral and Cellular Immune Responses to Inactivated Swine Influenza Virus Vaccine in Weaned Pigs

Humoral and cellular immune responses to inactivated swine influenza virus (SIV) vaccine were evaluated and compared. Fifty 3-week-old weaned pigs were randomly divided into the non-vaccinated control group and vaccinated group containing 25 pigs each. Pigs were vaccinated intramuscularly twice with adjuvanted UV-inactivated A/SW/MN/02011/08 (MN/08) H1N2 SIV vaccine at 6 and 9 weeks of age. Whole blood samples for multi-parameter flow cytometry (MP-FCM) and serum samples for hemagglutination inhibition (HI) assay were collected at 23 and 28 days after the second vaccination, respectively. A standard HI assay and MP-FCM were performed against UV-inactivated homologous MN/08 and heterologous pandemic A/CA/04/2009 (CA/09) H1N1 viruses. While the HI assay detected humoral responses only to the MN/08 virus, the MP-FCM detected strong cellular responses against the MN/08 virus and significant heterologous responses to the CA/09 virus, especially in the CD4+CD8+ T cell subset. The cellular heterologous responses to UV-inactivated virus by MP-FCM suggested that the assay was sensitive and potentially detected a wider range of antigens than what was detected by the HI assay. Overall, the adjuvanted UV-inactivated A/SW/MN/02011/08 H1N2 SIV vaccine stimulated both humoral and cellular immune responses including the CD4−CD8+ T cell subset

Albumin-conjugated PEG liposome enhances tumor distribution of liposomal doxorubicin in rats

To evaluate the effect of coupling of recombinant human serum albumin (rHSA) onto the surface of poly(ethylene glycol)-modified liposorne (PEG liposome) on the in vivo disposition characteristics of liposomal doxorubicin (DXR), the pharmacokinetics and tissue distribution of DXR were evaluated after intravenous administration of rHSA-modified PEG (rHSA/PEG) liposomal DXR into tumor-bearing rats. rHSA/PEG liposome prepared using a hetero-bifunctional cross-linker, N- succinimidyl 3-(2-pyridyldithio) propionate (SPDP), efficiently encapsulated DXR (over 95%). rHSA/PEG liposomal DXR showed longer blood-circulating property than PEG liposornal DXR and the hepatic and splenic clearances of rHSA/PEG liposornal DXR were significantly smaller than those of PEG liposomal DXR. It was also demonstrated that the disposition of DXR to the heart, one of the organs for DXR-related side-effects, was significantly smaller than free DXR. Furthermore, the tumor accumulation of rHSA/PEG liposomal DXR was significantly larger than that of PEG liposomal DXR. The &#34;therapeutic index&#34;, a criterion for therapeutic outcome, for rHSA/PEG fiposornal DXR was significantly higher than PEG liposomal DXR. These results clearly indicate that rHSA-conjugation onto the surface of PEG liposome would be a useful approach to increase the effectiveness and safety of PEG liposomal DXR.</p